Compositions and methods for cancer diagnosis

ABSTRACT

The present disclosure provides compositions comprising a food product, non-pathogenic microorganism, kits, methods of diagnosing a tumor in a subject, methods of quantifying the number of cancer cells in a cell sample, and methods of detecting a cancer cell, cancer tissue, or cell associated with a hyperproliferative disorder. In some embodiments, the method comprises a step of detecting the presence or absence of a modified substrate or portion thereof in urine of an animal without an instrument and solely by visual inspection of the urine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/776,689, filed Sep. 14, 2015, which in turn claims priorityto and is a United States National Stage filing under 35 U.S.C. 371 ofInternational PCT Application Serial No. PCT/US2014/030908, filed Mar.17, 2014, which claims priority to U.S. Provisional Application Nos.61/802,352, filed Mar. 15, 2013, and 61/832,147, filed Jun. 6, 2013,each of which is incorporated herein by reference in its entirety.

FIELD

The present disclosure is directed, in part, to compositions comprisinga non-pathogenic microorganism, kits, methods of diagnosing a tumor in asubject, methods of quantifying the number of cancer cells in a cellsample, and methods of detecting a cancer cell, cancer tissue, or cellassociated with a hyperproliferative disorder.

BACKGROUND

Current diagnosis of cancer typically requires instrumentation andcostly technology involving expensive reagents and long wait times toobtain results. The expanding field of synthetic biology has yet to befully leveraged for clinical applications, primarily due to safetyconcerns and a difficulty in engineering robust circuits in vivo.Accelerating high impact medical applications require utilizing methodsthat interface directly with medical infrastructure, genetic circuitsthat function outside of the controlled lab setting, and safe andclinically-accepted microbial hosts. Bacteria have been geneticallyengineered to participate in luminescence-, PET-, and MR1-based imagingmodalities for tumor detection [CITES]. Although these diagnostics eachhave specific utility, they have required intravenous or intratumoraldelivery and expensive equipment, limiting their application to selectcases. Strategies to employ quick and non-invasive diagnostic methodsfor cancer detection are desirable.

SUMMARY

The present disclosure provides compositions comprising a non-pathogenicmicroorganism comprising: a first nucleic acid sequence encoding anenzyme, or functional fragment thereof; a second nucleic acid sequenceencoding an polarization protein or proteins, or functional fragmentsthereof; and a third nucleic acid sequence encoding a toxin/antidotecombination; wherein the enzyme, or functional fragment thereof,catalyzes excitation of a portion of a substrate or the release of aportion of a substrate when the substrate is present in the blood of asubject; and wherein the polarization protein or proteins facilitatesthe inclusion of both the first and second nucleic acid sequences in thenon-pathogenic microorganism and its progeny upon cellular division.

The present disclosure also provides kits comprising the non-pathogenicmicroorganism compositions described herein.

The present disclosure provides methods of diagnosing a tumor in asubject comprising: i) administering a non-pathogenic microorganismcomposition described herein to a subject; ii) administering a substratefor the enzyme, or functional fragment thereof, to the subject; and iii)detecting the presence or absence of the released or excited portion ofthe substrate in the urine of the subject.

The present disclosure also provides methods of quantifying the numberof cancer cells in a cell sample comprising: i) contacting the cellsample with a non-pathogenic microorganism composition described hereinto form a mixture; ii) exposing the mixture to a substrate for theenzyme, or functional fragment thereof; and iii) measuring the amount ofthe released or excited portion of the substrate in the mixture.

The present disclosure also provides methods of detecting a cancer cell,cancer tissue, or cell associated with a hyperproliferative disorder ina subject comprising: i) administering non-pathogenic microorganismcomposition described herein to the subject; ii) administering asubstrate for the enzyme, or functional fragment thereof, to thesubject; and iii) detecting the presence or absence of the releasedportion of the substrate.

The present disclosure also provides any one or more of the foregoingnon-pathogenic microorganism compositions described herein for detectingthe presence of a tumor in a subject.

The present disclosure also provides any one or more of the foregoingnon-pathogenic microorganism compositions described herein for use inthe manufacture of a product for detecting the presence of a tumor in asubject.

The present disclosure also provides uses of any one or more of theforegoing non-pathogenic microorganism compositions described herein fordetecting the presence of a tumor in a subject.

The present invention also provides uses of any one or more of theforegoing non-pathogenic microorganism compositions described herein inthe manufacture of a medicament for detecting the presence of a tumor ina subject.

The present invention also provides a composition comprising anon-pathogenic microorganism comprising: one or more plasmids thatcomprise: a first nucleic acid sequence encoding an enzyme, orfunctional fragment thereof; a second nucleic acid sequence encoding atleast one polarization protein, or functional fragment thereof; and athird nucleic acid sequence encoding a combination of a toxin and anantidote; wherein the enzyme, or functional fragment thereof, processesa substrate into one or more detectable products when the substrate ispresent in the blood of a subject; and wherein the polarization protein(or, optionally, the combination of the polarization protein, toxin andan antidote) facilitates the inclusion of both the first and secondnucleic acid sequences in the non-pathogenic microorganism and itsprogeny upon cellular division. In some embodiments, the one or moreplasmids comprise the first nucleic acid sequence, the second nucleicacid sequence and the third nucleic acid sequence. In some embodiments,the invention relates to any of the compositions disclosed herein,wherein the enzyme, or functional fragment thereof, catalyzes excitationof a portion of a substrate or the release of a portion of a substratewhen the substrate is present in the blood of a subject.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that is a bacterium.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that is a Gram-negative bacterium.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that is chosen from: Escherichia spp.,Firmicutes spp., Bacteroidetes spp., Lactobacillus spp., Bifidobacteriaspp., or Acidopholus spp.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that is E. coli Nissle 1917 (EcN).

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that is harvested from a human or animalsample.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that comprises a first nucleic acidsequence encoding an enzyme, or functional fragment thereof, wherein theenzyme, or functional fragment thereof, cleaves a portion of asubstrate. In some embodiments, the enzyme, or functional fragmentthereof, is beta-galactosidase or a functional fragment ofbeta-galactosidase.

In some embodiments, any of the compositions disclosed herein comprise anon-pathogenic microorganism that comprises a first nucleic acidsequence encoding an enzyme, or functional fragment thereof, wherein theenzyme, or functional fragment thereof excites a substrate therebyemitting a detectable wavelength of light. In some embodiments, theenzyme, or functional fragment thereof processes a substrate into areaction product capable of emitting a detectable wavelength of lightupon exposure to a wavelength of light sufficient for excitation of thereaction product. In some embodiments, the reaction product of theenzyme processing the substrate is capable of being excreted in urinewhen the substrate and enzyme are administered to animal. In someembodiments, the reaction product of the enzyme processing the substrateis capable of being excreted in urine when the substrate and enzyme areadministered to animal per os. In some embodiments, the reaction productof the enzyme processing the substrate is capable of being excreted inurine when either the substrate or the enzyme are administered to animalper os. In some embodiments, the reaction product of the enzymeprocessing the substrate is capable of being excreted in urine when thesubstrate is administered intravenously and the enzyme are administeredto animal per os as a nucleic acid sequence.

The invention relates to any of the compositions disclosed hereincomprising any of the non-pathogenic microorganism disclosed hereinwherein the substrate, portion of substrate or the reaction productemits a visible wavelength of light when exposed to white light.

The invention relates to any of the compositions disclosed hereincomprising any of the non-pathogenic microorganism disclosed herein,wherein the polarization protein is a cytoplasmic microtubule associatedprotein. In some embodiments, the nucleic acid that encodes apolarization protein encodes AlpA or a functional fragment thereof. Insome embodiments, the nucleic acid that encodes a polarization proteinencodes an actin-like protein A (AlpA) family member or a functionalfragment thereof. In some embodiments, the nucleic acid that encodes apolarization protein encodes AlpA7 or a functional fragment thereof thathas at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentify to AlpA7. The invention relates to any of the compositionsdisclosed herein comprising any of the non-pathogenic microorganismdisclosed herein, wherein the non-pathogenic microorganism islyophilized, dried in a powder, or other solid dosage form. In someembodiments, the non-pathogenic microorganism is in a liquid dosageform.

The present invention also relates to kits comprising any of thecomposition disclosed herein. In some embodiments, the kits furthercomprise a substrate in place of an enzyme, or functional fragmentthereof. In some embodiments, the kits further comprise at least a firstcontainer comprising a rehydration solution and, optionally, a syringeand/or needle.

The invention further provides a method of diagnosing or detecting thepresence of a tumor in a subject comprising: i) administering anycomposition disclosed herein, ii) administering a substrate, orfunctional fragment thereof, for the enzyme, or functional fragmentthereof, to the subject wherein the enzyme produced by the microorganismin or on a tumor cell converts the substrate into one or more productsthat are excreted in urine; iii) detecting in the urine of the subject,the presence or absence of one or more products produced by theprocessing of the substrate by the enzyme. The invention furtherprovides any of the methods disclosed herein, wherein one or moreproducts produced by the processing of the substrate by the enzyme (eacha reaction product) can be visually detected by color in the urine ofthe subject.

The invention further provides any of the methods disclosed herein,wherein the one or more products produced by the processing of thesubstrate by the enzyme can be detected in the urine of the subject bycombining a sample of the urine with a reactant to produce a detectableresult not produced when a sample of urine free of the product iscombined with the reactant.

The invention further provides any of the methods disclosed herein,wherein the one or more products produced by the processing of thesubstrate by the enzyme is the released or excited portion of thesubstrate.

The invention further provides any of the methods disclosed herein,wherein any of the compositions disclosed herein is or are administeredto the subject per os. In some embodiments, the compositions disclosedherein are not administered to the subject per intravenous injection,per subcutaneous injection, or per intramuscular injection. In someembodiments, the compositions disclosed herein are not administered tothe subject per injection. The invention further provides any of themethods disclosed herein, wherein any of the compositions disclosedherein is or are administered to the subject per intravenous injection.

The invention further provides any of the methods disclosed herein,wherein the presence or absence of the one or more released or excitedportions of the substrate (or one or more reaction products) isdetermined by identifying a change in the color of the urine.

The invention further provides any of the methods disclosed herein,wherein the substrate is chosen from S-Gal, Ch-Red, and LuGal.

The invention further provides any of the methods disclosed herein,further comprising allowing a period of time to elapse after step i)sufficient for colonization of the microorganism in a tumor cell, tumortissue, or a cell associated with a hyperproliferative disorder. In someembodiments, the period of time to elapse after step (i) is no more thanabout 72 hours. In some embodiments, the period of time to elapse afterstep (i) is no more than about 48 hours. In some embodiments, the periodof time to elapse after step (i) is no more than about 36 hours. In someembodiments, the period of time to elapse after step (i) is no more thanabout 24 hours.

The invention further provides any of the methods disclosed herein,wherein, when the substrate is SGal, the presence or absence of thereleased or excited portion of the substrate is determined by contactinga urine sample from the subject to iron ions.

The invention further provides any of the methods disclosed herein,wherein when the substrate is D-luciferin-O-β-galactopyranoside (LuGal),the presence or absence of the released or excited portion of thesubstrate is determined by quantifying the amount of luciferin in theurine sample of the subject.

The invention further provides any of the methods disclosed herein,wherein the tumor is derived from the gastrointestinal tract or urinarysystem of the subject.

The invention further provides any of the methods disclosed herein,wherein the one or more products produced by the processing of thesubstrate by the enzyme is detected by comparing the amount of productin the urine of the subject to a background level, wherein a backgroundlevel is the amount of product that would be found in the urine of atumor free control subject undergoing the method.

The invention also provides a method of quantifying the number of cancercells in a cell sample comprising: i) contacting the cell sample withany of the compositions disclosed herein to form a mixture; ii) exposingthe mixture to a substrate for the enzyme, or functional fragmentthereof; and iii) measuring the amount of the released or excitedportion of the substrate in the mixture, or the amount of one or morereaction products.

The invention also provides any of the methods disclosed herein, whereinthe quantity of the one or more released or excited portions of thesubstrate or the one or more reaction products is determined bymeasuring luminescence, fluorescence or emission of visible wavelengthsof light when exposed to white light. The invention also provides any ofthe methods disclosed herein, wherein the quantity of the one or morereleased or excited portions of the substrate or the one or morereaction products is determined by colorimetric analysis.

The invention also provides a method of detecting a cancer cell, cancertissue, or cell associated with a hyperproliferative disorder in asubject comprising: i) administering any the compositions disclosedherein to a subject; ii) administering a substrate for the enzyme, orfunctional fragment thereof, to the subject; iii) detecting the presenceor absence of one or more released or excited portions of the substrateor one or more reaction products, wherein the reaction is the reactionbetween the enzyme and the substrate.

The invention also provides a method of detecting a cancer cell, cancertissue, or cell associated with a hyperproliferative disorder in asubject comprising: i) administering any the compositions disclosedherein to a subject; ii) administering a substrate for the enzyme, orfunctional fragment thereof, to the subject; iii) detecting the presenceor absence of one or more released or excited portions of the substrateor one or more reaction products. In some embodiments, the inventionprovides a method of detecting a cancer cell, cancer tissue, or cellassociated with a hyperproliferative disorder in a subject comprising:i) administering any the compositions disclosed herein to a subject; ii)administering a substrate for the enzyme, or functional fragmentthereof, to the subject; iii) detecting the presence or absence of oneor more released or excited portions of the substrate or one or morereaction products, wherein the composition is administered to thesubject per os. In some embodiments, the invention provides a method ofdetecting a cancer cell, cancer tissue, or cell associated with ahyperproliferative disorder in a subject comprising: i) administeringany the compositions disclosed herein to a subject; ii) administering asubstrate for the enzyme, or functional fragment thereof, to thesubject; iii) detecting the presence or absence of one or more releasedor excited portions of the substrate or one or more reaction products,wherein the composition is administered to the subject per intravenousinjection. In some embodiments, the invention provides a method ofdetecting a cancer cell, cancer tissue, or cell associated with ahyperproliferative disorder in a subject comprising: i) administeringany the compositions disclosed herein to a subject; ii) administering asubstrate for the enzyme, or functional fragment thereof, to thesubject; iii) detecting the presence or absence of one or more releasedor excited portions of the substrate or one or more reaction products,wherein the composition is administered to the subject per os and thesubstrate is administered per os. In some embodiments, the inventionprovides a method of detecting a cancer cell, cancer tissue, or cellassociated with a hyperproliferative disorder in a subject comprising:i) administering any the compositions disclosed herein to a subject; ii)administering a substrate for the enzyme, or functional fragmentthereof, to the subject; iii) detecting the presence or absence of oneor more released or excited portions of the substrate or one or morereaction products, wherein the composition is administered to thesubject per os and the substrate is administered per intravenousinjection. In some embodiments, the invention provides a method ofdetecting a cancer cell, cancer tissue, or cell associated with ahyperproliferative disorder in a subject comprising: i) administeringany the compositions disclosed herein to a subject; ii) administering asubstrate for the enzyme, or functional fragment thereof, to thesubject; iii) detecting the presence or absence of one or more releasedor excited portions of the substrate or one or more reaction products,wherein the presence or absence of the one or more released or excitedportions of the substrate or the one or more reaction products isdetermined by quantifying the amount of released or excited portion ofthe substrate present in the subject at one or a plurality of sites inthe subject or by quantifying the amount of one or more reactionproducts present in the subject at one or a plurality of sites in thesubject.

In some embodiments, the invention provides a method of detecting acancer cell, cancer tissue, or cell associated with a hyperproliferativedisorder in a subject comprising: i) administering any the compositionsdisclosed herein to a subject; ii) administering a substrate for theenzyme, or functional fragment thereof, to the subject; iii) detectingthe presence or absence of one or more released or excited portions ofthe substrate or one or more reaction products, wherein the methodfurther comprises allowing a period of time to elapse after step i)sufficient for colonization of the microorganism in a cancer cell,cancer tissue, or a cell associated with hyperproliferative disorder. Insome embodiments, the invention provides a method of detecting a cancercell, cancer tissue, or cell associated with a hyperproliferativedisorder in a subject comprising: i) administering any the compositionsdisclosed herein to a subject; ii) administering a substrate for theenzyme, or functional fragment thereof, to the subject; iii) detectingthe presence or absence of one or more released or excited portions ofthe substrate or one or more reaction products, wherein the methodfurther comprises allowing a period of time to elapse after step i)sufficient for exponential growth of the microorganism in a cancer cell,cancer tissue, or a cell associated with hyperproliferative disorder.

In some embodiments, the invention provides a method of detecting acancer cell, cancer tissue, or cell associated with a hyperproliferativedisorder in a subject comprising: i) administering any the compositionsdisclosed herein to a subject; ii) administering a substrate for theenzyme, or functional fragment thereof, to the subject; iii) detectingthe presence or absence of one or more released or excited portions ofthe substrate or one or more reaction products, and optionally furthercomprising allowing a period of time to elapse after step i) sufficientfor colonization of the microorganism in a cancer cell, cancer tissue,or a cell associated with hyperproliferative disorder; wherein, when thesubstrate is SGal, the presence or absence of one or more released orexcited portions of the substrate is determined after administration ofa composition comprising iron ions.

In some embodiments, the invention provides a method of detecting acancer cell, cancer tissue, or cell associated with a hyperproliferativedisorder in a subject comprising: i) administering any the compositionsdisclosed herein to a subject; ii) administering a substrate for theenzyme, or functional fragment thereof, to the subject; iii) detectingthe presence or absence of one or more released or excited portions ofthe substrate or one or more reaction products, and optionally furthercomprising allowing a period of time to elapse after step i) sufficientfor colonization of the microorganism in a cancer cell, cancer tissue,or a cell associated with hyperproliferative disorder; wherein thecancer cell, cancer tissue or cell associated with a hyperproliferativedisorder is a cancer cell or tissue derived from the gastrointestinaltract or urinary system of a subject. In some embodiments, the inventionprovides any of the methods disclosed herein, wherein the step ofdetecting the presence or absence of one or more released or excitedportions of the substrate or one or more reaction products is determinedwithout the assistance or reliance or use of an instrument. In someembodiments, the invention provides any of the methods disclosed herein,wherein the step of detecting the presence or absence of one or morereleased or excited portions of the substrate or one or more reactionproducts is determined by visual colorimetric analysis of the human eye.

In some embodiments, the invention provides any of the methods disclosedherein, wherein the cancer cell, cancer tissue or cell associated with ahyperproliferative disorder is a metastatic cancer cell or cancertissue.

In some embodiments, the invention provides any of the methods disclosedherein, wherein the cancer cell, cancer tissue or cell associated with ahyperproliferative disorder is a metastatic cancer cell or cancer tissuepresent in the liver.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a non-limiting example of a plasmid containing thenucleic acid sequence encoding the enzyme (LacZ), the nucleic acidsequence encoding the polarization protein (AlpA), and the nucleic acidencoding the toxin-antidote combination (hok/sok).

FIG. 2A-2D depicts a summary of the diagnostic platform and accompanyingdata utilizing administration of a plasmid comprising the LacZ (2A) Theprobiotic urinary cancer diagnostic platform. EcN-lacZ is introducedsystemically and specifically colonizes liver metastases. One or morecommercially available substrates are injected, and activated substrateis measured in the urine. Application-specific substrates can bedetected via MRI, luminescence, or visual color change. (2B) In vivoimaging screen (IVIS) images demonstrating colonization of EcNcomprising nucleic acid sequences in liver metastases 24-hours followingoral administration of the non-pathogenic bacteria. (2C) Quantificationof activated product in the urine via luciferase assay for the EcN-lacZplatform. (2D) Instrument-free diagnosis of cancer metastasis by urinarycolor change.

FIG. 3A-3D depicts a set of experiments using ECN bacterial strainscomprising lacZ fed to mice. FIG. 3A demonstrates successfulcolonization of mice after 24 hours of administration. FIG. 3B depictsthe urine color change from mice fed bacterial compositions disclosedherein and subsequently administered with lacZ substrate as compared tourine from tumor-bearing mice without being fed the bacteria-containingcompositions. A color change indicates presence of our engineeredbacteria and presence of a tumor. FIG. 3C depicts an experiment in whichluminescence of urine is measure by a LuGal quantification assay allowsfor, yielding a 5-10× signal to noise ratio over control. FIG. 3Ddepicts an experiment in which uncleaved substrate levels are measuredand can be used as an additional control for determining the absence,presence or quantities of tumor cells in mice.

FIG. 4 depicts a schematic of how the method to tumor detection isconceptualized. Tumors are colonized by an engineered probiotic fedorally. Substrate is injected systemically and a cleavage productproduces a readily-detectable color change in the urine.

FIG. 5 depicts a schematic of the function of the hok/sok(toxin/antidote combination) in the upper panel. FIG. 5 lower panel alsodepicts an alternative system for plasmid pairing is used for increasedstability of plasmid in transformed bacterial strains. The parA locus ofplasmid R1 encodes a prokaryotic centromere-like system that mediatesgenetic stabilization of plasmids by an unknown mechanism. The locuscodes for two proteins, ParM and ParR, and a centromere-like DNA region(parC) to which the ParR protein binds. ParR mediates specific pairingof parC-containing DNA molecules in vitro. ParM forms discrete foci thatlocalize to specific cellular regions in a simple, yet dynamic pattern.In newborn cells, ParM foci were present close to both cell poles.Concomitant with cell growth, new foci formed at mid-cell. A pointmutation that abolished the ATPase activity of ParM simultaneouslyprevented cellular localization and plasmid partitioning. AparA-containing plasmid localized to similar sites, i.e. close to thepoles and at mid-cell, thus indicating that the plasmid co-localizeswith ParM. Previous data show that plasmid DNA and ParM co-localize andthat parA is a true partitioning system that mediates pairing ofplasmids at mid-cell and subsequently moves them to the cell polesbefore cell division.

FIG. 6 depicts a measure of colony-forming units in cells transformedwith hok/sok nucleic acid sequences versus cell transformed with thehok/sok nucleic acid sequences as well as the alpA nucleic acidsequence. The number of colonies in subjects administered with the bothset of bacteria had similar growth rates.

FIG. 7 depicts the stability of transformed plasmid in compositionscomprising the bacteria of the present invention over a time period of24 and 72 hours.

FIG. 8 depicts a schematic of the function of the PROP-Z diagnosticplatform. The PROP-Z diagnostic platform is made up of probiotic E. coliNissle 1917 (EcN) bacteria transformed with a dual-stabilized, highexpression lacZ vector. (1) PROP-Z is delivered orally. (2) Bacteriarapidly (24 hours) translocate across the GI Tract and (3) specificallyamplify within metastatic tumors in the liver. (4) PROP-Z express highlevels of the lacZ enzymatic marker, enabling urinary detection viainjected cleavable substrates.

FIG. 9A-9H depicts a summary of the dual-stabilized vector thatefficiently maintains diagnostic activity in vivo. (9A) Constitutive,inducible, and quorum sensing plasmid constructs were tested in vivousing a genetically. encoded luciferase as a proxy for diagnosticactivity. Inducible circuits were based on the lux promoter, where AHLwas added to the drinking water at a concentration of 10 uM. For quorumsensing constructs, autonomous activation by the AHL synthase LuxI wasquenched with the addition of 10 uM (Z.)4-Bromo-5-(bromomethylene)-2(5H)-furanone to the drinking water. (9B)Dual. stabilized maintenance system ensures total plasmid stability inthe tumor environment. Hok is a toxin antitoxin system that killsdaughter cells in the event of plasmid loss. Alp7 is a B. subtilisderived plasmid partitioning system that efficiently segregates plasmidsto cell poles during division. 9(C) To quantify the performance of ourmaintenance systems in vitro, we successively subcultured PROP. Zbacteria daily and compared colony counts (black/white colonies on LB S.Gal plates (SI). We found nearly 100% maintenance over 72 hours with thecombination of hok/sok and alp7CAR (means+--- s.e.m, n=4 each). (9D)Enzymatic activity was also maintained over 72 hours in vitro. (9E) Toquantify the performance of our PROP. Z diagnostic in stringent tumormicroenvironment conditions, we performed growth assays under lownutrient (varying glucose %), pH, and oxygen conditions. (9F) Inaddition to growth, enzymatic activity was maintained by thedually-stabilized vector. (9G) Further, to quantify in vivo maintenance,we injected PROP-⋅Z bacteria intravenously in a subcutaneous model ofcancer. Tumors were homogenized daily and analyzed by colony countratios. Plasmids were quickly lost in the absence of maintenance, hokenabled stability for 24 hours, and the combination of hok and alp7ensure total stability for 72 hours (means+--- s.e.m, n=5 each). (911)To measure PROP-Z diagnostic amplification due to EcN growth and lacZenzymatic turnover, we analyzed tumor homogenates taken in (c) for lacZactivity, finding a total in vivo amplification of around 5 for therelative cleavage rate from Day 3 to Day 0 (means+-- s.e.m, n=5 each).

FIG. 10A-10E depicts liver models of metastasis used to test theperformance of the diagnostic on the early detection of small tumorsdifficult to detect using traditional methods. (10A) Healthy (left) andmetastatic livers (right), showing small tumor nodules. (10B) 24 hoursfollowing oral administration, PROP-Z bacteria were detected in tumornodules via IVIS measurement. (10C) PROP-⋅Z bacteria colonize the innercore of metastatic nodules are often not visible from the exterior(left) but are revealed by cutting to the interior (right). Dashed line(left) indicates cut site. (10D) To determine safety and specificity, wecolony counted PROP-7 in organs following oral administration. Weobserved 106 bacteria colonize tumors after 24 hours with zerocolonization (0 CFU/g) in off. target organs and no growth over time(means+-- s.e.m, n=4 each). (10E) PROP-Z bacteria detect the presence ofmetastatic tumors via tumor specific growth leading to high-levelexpression of lacZ. We quantified enzymatic activity by injectingLu-gal, a lacZ substrate that when cleaved produces luciferin, andassayed the urine for luminescence (means+--- s.e.m, n=6 for eachcondition, 2 tail students p<0.05).

FIG. 11A-11B depicts a qPCR calculation of the number of bacteriapresent in the tumor sample. (11A) E. coli Nissle DNA samples were DNAsamples were diluted in DNA-free water and DNA was purified as above.Automated Ct values generated from a Bio-RAD iCycler machine showing alinear correlation with Ct value across several orders of magnitude.(11B) The number of bacteria were calculated using a semi-log regressionfit and the calculated #of bacteria is plotted in b, showing asensitivity of detection of around −100 bacteria per sample. Sampleswere repeated in quadruplicate, standard error shown is smaller than thepoints.

FIG. 12 depicts colony forming units with hok and hok+alpA stabilitysystems. The absolute levels of bacteria in tumors in our athymiccolorectal cancer model were measured using colonycounting methods. Thegrowth rates of both strains appear similar, suggesting that thestability of the combined system is not due to slower growth (in fact itgrows slightly faster and thus stability results would likely beunderestimated).

FIG. 13A-13B depicts growth and activity of N-ColoniZe platform indifferent oxygen, inducer, and antibiotic conditions. (13A) OD600 valuesare shown in varying oxygen conditions after 48 hours of growth(subcultured at 24 hours) in 14 mL Falcon tubes capped or uncapped withhigh level of media (14 mL) or low level of media (3 mL). (13B) MeasuredlacZ activity values in the same conditions. Norm indicates EZ-Richmedia with 30 0.2% glucose, 3 mL of media, and uncapped. Kanamycingrowth conditions for the cap/high case were not tested in either panel.

DETAILED DESCRIPTION OF EMBODIMENTS

Some embodiments relate to non-pathogenic microorganisms which compriseheterologous nucleic acid sequences. The non-pathogenic microorganismscolonize tumors. In some embodiments, the non-pathogenic microorganismsare non-pathogenic bacteria.

Some embodiments provide compositions comprising a non-pathogenicmicroorganism that comprise one or more plasmids, a nucleic acidsequence that encode a polarization protein, or functional fragmentthereof and a nucleic acid sequence that encode a combination of a toxinand an antidote. When such microorganism reproduce in vivo, such as whenthey are colonizing a tumor in an animal, a greater proportion of theresulting progeny remain plasmid-bearing compared to the proportion ofresulting progeny from microorganisms which do not comprise a nucleicacid sequence that encodes a polarization protein, or functionalfragment thereof and/or a nucleic acid sequence that encode acombination of a toxin and an antidote. The polarization protein, oroptionally, the combination of the polarization protein and a toxin andan antidote, reduces plasmid loss that occurs during cell division andfacilitates the inclusion of both the plasmids in the non-pathogenicmicroorganism and its progeny upon cellular division. This feature ofimproves diagnostics and therapeutics that use microorganisms thattarget and colonize tumors and release therapeutics or products involvedin tumor detection. In some embodiments, the microorganisms furthercomprise a nucleic acid sequence that encode a protein used intherapeutic methods that cause slowed or inhibit tumor growth or reduceor eliminate tumors. In some embodiments, the microorganisms furthercomprise a nucleic acid sequence that encode a protein used indiagnostic methods. The protein may be a marker which can be detecteddirectly or indirectly. In some embodiments, the marker is an enzymewhich when produced by the microorganism can process a substrate such asa substrate delivered to the animal by injection. In some embodiments,the marker is an substrate which, when produced by the microorganism,can be processed by an enzyme such as an enzyme delivered to the animalby injection. In some embodiments, the marker is a protein which can bedetected directly.

Some embodiments provide compositions comprising a non-pathogenicmicroorganism that comprise one or more plasmids, and a nucleic acidsequence that encode a protein which is used to produce a detectableindicator in the urine of an animal. In some embodiments, the protein isan enzyme which can convert a substrate into detectable products. Insome embodiments, substrate is injected in the animal such as byinjection into the circulatory system (intrarterial or Intravenously(IV)), subcutaneous (SC) injection or intramuscular (IM) injection orother parenteral injection or by oral administration. In someembodiments, the protein is an enzyme which can convert a substrate intodetectable products that are excreted in the urine of the animal. Insome embodiments, the protein is an enzyme which can convert a substrateinto colored products that are excreted in the urine of the animal andcan be detected by visual inspection of the animal's urine. In someembodiments, the protein is an enzyme which can convert a detectablesubstrate into undetectable products. In some embodiments, enzyme isinjected in the animal such as by injection into the circulatory system(intrarterial or IV), SC injection or IM injection or other parenteralinjection or by oral administration. In some embodiments, the protein isan substrate which can be converted into a detectable product or areaction product. In some embodiments, the protein is an substrate whichcan be converted by an enzyme into a detectable product that is excretedin the urine of the animal. In some embodiments, the protein is asubstrate that can be converted by an enzyme into colored products thatare excreted in the urine of the animal and can be detected by visualinspection of the animal's urine. In some embodiments, themicroorganisms further comprise a nucleic acid sequence that encodes apolarization protein, or functional fragment thereof, and a nucleic acidsequence that encodes a combination of a toxin and an antidote, orfunctional fragments thereof.

Some embodiments provide methods of detecting tumors in an subject. Thenon-pathogenic microorganisms which comprise a nucleic acid sequencethat encodes a protein which is used to produce a detectable indicatorin the urine of an animal as administered to the animal. Thenon-pathogenic microorganisms may be bacteria. The non-pathogenicmicroorganisms may be Gram-negative bacteria. The non-pathogenicmicroorganisms may be administered orally. The non-pathogenicmicroorganisms may be administered orally, by solid dosage form orliquid dosage form, in either case, optionally as part of a foodproduct. The non-pathogenic microorganisms colonize tumors, or groups ofcancer cells. In some embodiments, the tumors or groups of cancer cellsare of the gastrointestinal (GI) track. In some embodiments, the tumorsor groups of cancer cells are GI-derived metastasic tumors. In someembodiments, the tumors or groups of cancer cells are primary non-GIderived tumors. In some embodiments, the tumors or groups of cancercells are metastatic non-GI derived tumors.

After a sufficient amount of time elapses for colonization to proceed,the urine of the subject is analyzed to determine the present of thedetectable indicator in the urine of a subject. In some embodiments, thedetectable indicator is a detectable product produced by processing ofthe substrate by an enzyme encoded by nucleic acid sequences in themicroorganism. In such instances, the unprocessed substrate isadministered to the subject prior to collecting urine from the subject.

There are several advantages of current technology compared to existingdiagnostic agents: (1) in some embodiments, color changes are readilyobservable to the eye so no instrumentation is required, thus enablingrapid-response and field applications; and (2) Since administration isdone with a probiotic that naturally colonizes human gut flora, safetyhas already been well established. Additionally, probiotics areroutinely sold to the public for consumption, opening the possibilityfor wider applicability in home use. (3) Bacteria, unlike nanoparticleagents, replicate to levels of 10⁶-10⁹ rapidly over the course of 2 daysleading to amplification of signal. Additionally production of an enzymewhich can cleave multiple substrates leads to an added level ofamplification. (4) To date, no technologies exist that attempt to detecttumors via urine. Substantial existing medical infrastructure forurinalysis could therefore be blended with this technology. (5) Thesystem is essentially free to produce, as bacterial media is the onlynecessary material. The probiotic compositions are useful for methodsfor detection of solid tumors in a variety of subtypes. These can be GItract tumors that bacteria have easy access to or remote metastases thathave been shown to be colonizable. In addition, this platform allows formany imaging modalities including PET, MRI, and luminescent detection tobe used simultaneously. This work provides a significant decrease incost with a simultaneous increase in flexibility in terms of diagnosticcapabilities.

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

The term “active state” refers to the conformation or set ofconformations of a polypeptide that allows functional domain or domainsof the polypeptide to associate or disassociate with another compound,macromolecule, or ligand. In some embodiments, the association ordisassociation of the polypeptide with another compound, macromolecule,or ligand may propagate or inhibit a biological signal. In someembodiments, the polypeptide is an enzyme with a catalytic site orfunctional domain that binds or associates to a substrate upon adoptinga conformation or set of conformations in the enzyme's one or moreactive states.

The terms “amino acid” refer to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon. Suitable amino acids include, without limitation, both the D-and L-isomers of the naturally-occurring amino acids, as well asnon-naturally occurring amino acids prepared by organic synthesis orother metabolic routes. In some embodiments, a single “amino acid” mighthave multiple sidechain moieties, as available per an extended aliphaticor aromatic backbone scaffold. Unless the context specifically indicatesotherwise, the term amino acid, as used herein, is intended to includeamino acid analogs including non-natural analogs.

The terms “functional fragment” means any portion of a polypeptide ornucleic acid sequence from which the respective full-length polypeptideor nucleic acid relates that is of a sufficient length and has asufficient structure to confer a biological affect that is similar orsubstantially similar to the full-length polypeptide or nucleic acidupon which the fragment is based. In some embodiments, a functionalfragment is a portion of a full-length or wild-type nucleic acidsequence that encodes any one of the nucleic acid sequences disclosedherein, and said portion encodes a polypeptide of a certain lengthand/or structure that is less than full-length but encodes a domain thatstill biologically functional as compared to the full-length orwild-type protein. In some embodiments, the functional fragment may havea reduced biological activity, about equivalent biological activity, oran enhanced biological activity as compared to the wild-type orfull-length polypeptide sequence upon which the fragment is based. Insome embodiments, the functional fragment is derived from the sequenceof an organism, such as a human. In such embodiments, the functionalfragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%sequence identity to the wild-type human sequence upon which thesequence is derived. In some embodiments, the functional fragment mayretain 85%, 80%, 75%, 70%, 65%, or 60% sequence homology to thewild-type sequence upon which the sequence is derived. The presentinvention also comprises functional fragments nucleotide sequences thatencode a polypeptide capable of enzymatic activity, substrate activity,polarization activity, toxin activity, or antidote to toxin activity asdisclosed herein in an animal. In some embodiments, the functionalfragment are DNA fragments selected from at least one of the variousnucleotide sequences disclosed herein, including SEQ ID NO: 1 and can beany of the following described DNA fragments, as it applies to thespecific encoding nucleic acid sequence provided herein. In someembodiments, DNA fragments can comprise 30 or more, 45 or more, 60 ormore, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 ormore, 480 or more, 540 or more, 600 or more, 660 or more, 720 or more,780 or more, 840 or more, 900 or more, 960 or more, 1020 or more, 1080or more, 1140 or more, 1200 or more, 1260 or more, 1320 or more, 1380 ormore, 1440 or more, 1500 or more, 1560 or more, 1620 or more, 1680 ormore, or 1740 or more, 1800 or more, 2000 or more, 2100 or more, 2200 ormore, 2300 or more, 2400 or more, 2500 or more, 2600 or more, 2700 ormore, 2800 or more, 2900 or more, 3000 or more, 4000 or more, 4500 ormore, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 ormore, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 ormore, 10000 or more, 10100 or more nucleotides. In some embodiments, DNAfragments can comprise coding sequences for partitioning proteins,toxin/antidote combinations, and enzymes disclosed herein. In someembodiments, DNA fragments can comprise fewer than 60, fewer than 75,fewer than 90, fewer than 120, fewer than 150, fewer than 180, fewerthan 210, fewer than 240, fewer than 270, fewer than 300, fewer than360, fewer than 420, fewer than 480, fewer than 540, fewer than 600,fewer than 660, fewer than 720, fewer than 780, fewer than 840, fewerthan 900, fewer than 960, fewer than 1020, fewer than 1080, fewer than1140, fewer than 1200, fewer than 1260, fewer than 1320, fewer than1380, fewer than 1440, fewer than 1500, fewer than 1560, fewer than1620, fewer than 1680, or fewer than 1740, fewer than 1800, fewer than1900, fewer than 2000, fewer than 2100, fewer than 2200, fewer than2300, fewer than 2400, fewer than 2500, fewer than 2600, fewer than2700, fewer than 2800, fewer than 2900, fewer than 3000, fewer than4000, fewer than 5000, fewer than 6000, fewer than 7000, fewer than8000, fewer than 9000, or fewer than 10000 nucleotides. In someembodiments, the functional fragments are nucleic acid fragments of SEQID NO:1 and include one or more nucleic acid derivatives. In someembodiments, the functional fragments are nucleic acid fragments of SEQID NO:1 and include more than 5, 10, 15, 20, 25, or 30 nucleic acidderivatives. In some embodiments, the functional fragments are nucleicacid fragments of SEQ ID NO:1 and include one or more conservativesubstitutions. In some embodiments, the fragments of the presentinvention comprise or possess at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% homology to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO: 10, or SEQ ID NO:11. In some embodiments, plasmids of theinvention comprise at least one or a combination of fragments of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% homology to SEQID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ IDNO:11, each of which possess the function of the full-length SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO:11respectively.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:1. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:1.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:2. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:2. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:2.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:3. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:3. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:3.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:4. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:4. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:4.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:5. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:5. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:5.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:6. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:6. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:6.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:7. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:7. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:7.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:8. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:8. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:8.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:9. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:9. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:9.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:01. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:10. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:10. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:10.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:11. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:11. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:11.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 70% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 75% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:12. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:12. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:12.

The terms “hyperproliferative disorder” refer to a disordercharacterized by abnormal proliferation, abnormal growth, abnormalsenescence, abnormal quiescence, or abnormal removal of cells any or inan organism, and includes include hyperplasias, neoplasias, cancer,fibroproliferative disorders (such as involving connective tissues, aswell as other disorders characterized by fibrosis, including forexample, rheumatoid arthritis, insulin dependent diabetes mellitus,glomerulonephritis, cirrhosis, and scleroderma), smooth muscleproliferative disorders (such as atherosclerosis and restinosis),chronic inflammation, and epithelial cell proliferative disorders (forexample, psoriasis; keratosis; acne; comedogenic lesions; verracouslesions such as verruca plana, plantar warts, verruca acuminata, andother verruciform lesions marked by proliferation of epithelial cells;folliculitis and pseudofolliculitis; keratoacanthoma; callosities;Darier's disease; ichfhyosis; lichen planus; molluscous contagiosum;melasma; Fordyce disease; and keloids or hypertrophic scars). In someembodiments, the hyperproliferative disease is a cancer derived from thegastrointestinal tract or urinary system. In some embodiments, ahyperproliferative disease is a cancer of the adrenal gland, bladder,bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia,gastrointestinal tract, stomach, colon, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments,the term hyperproliferative disease is a cancer chosen from: lungcancer, bone cancer, CMML, pancreatic cancer, skin cancer, cancer of thehead and neck, cutaneous or intraocular melanoma, uterine cancer,ovarian cancer, rectal cancer, cancer of the anal region, stomachcancer, colon cancer, breast cancer, testicular, gynecologic tumors(e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina orcarcinoma of the vulva), Hodgkin's disease, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system (e.g.,cancer of the thyroid, parathyroid or adrenal glands), sarcomas of softtissues, cancer of the urethra, cancer of the penis, prostate cancer,chronic or acute leukemia, solid tumors of childhood, lymphocyticlymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g.,renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms ofthe central nervous system (e.g., primary CNS lymphoma, spinal axistumors, brain stem gliomas or pituitary adenomas).

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence that encodes one or moreof the polypeptides. In some embodiments, the genetic construct encodesone or more of the polypeptides described herein. In some embodiments,the nucleotide sequence that encodes a one or more of the polypeptidesdescribed herein, or coding sequence, includes initiation andtermination signals operably linked to regulatory elements including abacterial promoter and polyadenylation signal capable of directingexpression in the cells of the bacteria in which the nucleic acidmolecule is transformed. In some embodiments, the composition orpharmaceutical compositions, kits or nucleic acids comprises at leastone genetic construct comprising at least one or combination of: SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9 or functional fragments of any disclosed percent homologydisclosed herein. In some embodiments, the composition or pharmaceuticalcompositions, kits or nucleic acids comprises at least one geneticconstruct comprising at least one or combination of: SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11 orfunctional fragments of any disclosed percent homology disclosed herein.In some embodiments, the composition or pharmaceutical compositions,kits or nucleic acids comprises at least one genetic constructcomprising at least one or combination of: SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11 or functionalfragments thereof with any disclosed percent homology disclosed herein,optionally comprising at least one or a combination of: SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO:9, or functional fragments thereof with anydisclosed percent homology disclosed herein. In some embodiments, thecomposition or pharmaceutical compositions, kits or nucleic acidscomprises at least one genetic construct comprising SEQ ID NO:5 or anyfunctional fragment thereof with any disclosed percent homologydisclosed herein.

The terms “effective amount” refers to an amount of a compound,material, or composition, as described herein effective to achieve aparticular biological result such as, but not limited to, biologicalresults disclosed, described, or exemplified herein. Such results mayinclude, but are not limited to colonization of a tumor byadministration of a composition disclosed herein, or production of anamount of a substrate, portion thereof, or a reaction product afterexposure of the substrate with an enzyme such that the presence, absenceor quantity of substrate, portion thereof, or a reaction productdetermined by any means suitable in the art. In some embodiments, thebiological result is an amount of a substrate, portion thereof, or areaction product after exposure of the substrate with an enzyme suchthat the presence, absence or quantity of substrate, portion thereof, ora reaction product determined by visual inspection of urine excreted bya subject administered any one or more compositions disclosed herein.The effective amount of the composition may be dependent on any numberof variables, including without limitation, the species, breed, size,height, weight, age, overall health of the subject, the type offormulation, the mode or manner of administration, the type and/orseverity of the particular condition being treated.

The terms “electroporation,” “electro-permeabilization,” or“electro-kinetic enhancement” (“EP”) as used interchangeably hereinrefer to the use of a transmembrane electric field pulse to inducemicroscopic pathways (pores) in a bio-membrane; their presence allowsbiomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, andwater to pass from one side of the cellular membrane to the other.

As used herein, the term “expressible form” refers to gene constructsthat contain the necessary regulatory elements operable linked to acoding sequence that encodes one or more proteins described herein, suchthat, when present in a transformed or transfected cell, the codingsequence will be expressed.

As used herein, the term “non-pathogenic microorganism” refers to amicroorganism that is not capable of causing a disease or disorder whenadministered to an animal, including a human. In some embodiments, themicroorganism is incapable of causing a disease or disorder whenadministered to a mammal. In some embodiments, the microorganism is anon-pathogenic microorganism incapable of causing a disease or disorderwhen administered to a human or domesticated animal (such as a dog, cat,horse, sheep, cow, goat, pig, etc.). In some embodiments, themicroorganism is non-pathogenic microorganism incapable of causing adisease or disorder when administered to a human. In some embodiments,the microorganism is non-pathogenic microorganism is chosen from any oneof the bacterial species identified in Table 1. In some embodiments, thenon-pathogenic microorganism is not one of species listed on Table 1. Insome embodiments, the non-pathogenic microorganism is an attenuatedbacterial strain. In some embodiments, the non-pathogenic microorganismis an attenuated bacterial strain listed on Table 1. In someembodiments, the non-pathogenic microorganism is an attenuated bacterialstrain genetically modified to silence, remove, or mutate a virulencefactor.

The term “nucleic acid” refers to a molecule comprising two or morelinked nucleotides. “Nucleic acid” and “nucleic acid molecule” are usedinterchangeably and refer to oligoribonucleotides as well asoligodeoxyribonucleotides. The terms also include polynucleosides (i.e.,a polynucleotide minus a phosphate) and any other organic basecontaining nucleic acid. The organic bases include adenine, uracil,guanine, thymine, cytosine and inosine. The nucleic acids may be singleor double stranded. The nucleic acid may be naturally or non-naturallyoccurring. Nucleic acids can be obtained from natural sources, or can besynthesized using a nucleic acid synthesizer (i.e., synthetic).Isolation of nucleic acids are routinely performed in the art andsuitable methods can be found in standard molecular biology textbooks.(See, for example, Maniatis' Handbook of Molecular Biology.) The nucleicacid may be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA,cDNA, rRNA, miRNA, PNA or LNA, or a combination thereof, as describedherein. Non-naturally occurring nucleic acids such as bacterialartificial chromosomes (BACs) can also be used in accordance with someaspects of this invention.

Some aspects of this invention relate to the use of nucleic acidderivatives. The use of certain nucleic acid derivatives may increasethe stability of the nucleic acids of the invention by preventing theirdigestion, particularly when they are exposed to biological samples thatmay contain nucleases. As used herein, a nucleic acid derivative is anon-naturally occurring nucleic acid or a unit thereof. Nucleic acidderivatives may contain non-naturally occurring elements such asnon-naturally occurring nucleotides and non-naturally occurring backbonelinkages. Nucleic acid derivatives according to some aspects of thisinvention may contain backbone modifications such as but not limited tophosphorothioate linkages, phosphodiester modified nucleic acids,combinations of phosphodiester and phosphorothioate nucleic acid,methylphosphonate, alkylphosphonates, phosphate esters,alkylphosphonothioates, phosphoramidates, carbamates, carbonates,phosphate triesters, acetamidates, carboxymethyl esters,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof. The backbone composition of the nucleic acids may behomogeneous or heterogeneous. Nucleic acid derivatives according to someaspects of this invention may contain substitutions or modifications inthe sugars and/or bases. For example, some nucleic acid derivatives mayinclude nucleic acids having backbone sugars which are covalentlyattached to low molecular weight organic groups other than a hydroxylgroup at the 3′ position and other than a phosphate group at the 5′position (e.g., an 2′-0-alkylated ribose group). Nucleic acidderivatives may include non-ribose sugars such as arabinose. Nucleicacid derivatives may contain substituted purines and pyrimidines such asC-5 propyne modified bases, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouraciland pseudoisocytosine. In some embodiments, a nucleic acid may comprisea peptide nucleic acid (PNA), a locked nucleic acid (LNA), DNA, RNA, ora co-nucleic acids of the above such as DNA-LNA co-nucleic acid.

As used herein the term “isolated nucleic acid molecule” refers to anucleic acid that is not in its natural environment, for example anucleic acid that has been (i) extracted and/or purified from a cell ormicrobe, for example, a bacteria or yeast, by methods known in the art,for example, by alkaline lysis of the host cell and subsequentpurification of the nucleic acid, for example, by a silica adsorptionprocedure; (ii) amplified in vitro, for example, by polymerase chainreaction (PCR); (iii) recombinantly produced by cloning, for example, anucleic acid cloned into an expression vector; (iv) fragmented and sizeseparated, for example, by enzymatic digest in vitro or by shearing andsubsequent gel separation; or (v) synthesized by, for example, chemicalsynthesis. In some embodiments, the term “isolated nucleic acidmolecule” refers to (vi) an nucleic acid that is chemically markedlydifferent from any naturally occurring nucleic acid. In someembodiments, an isolated nucleic acid can readily be manipulated byrecombinant DNA techniques well known in the art.

Accordingly, a nucleic acid cloned into a vector, or a nucleic aciddelivered to a host cell and integrated into the host genome isconsidered isolated but a nucleic acid in its native state in itsnatural host, for example, in the genome of the host, is not. Anisolated nucleic acid may be substantially purified, but need not be.For example, a nucleic acid that is isolated within a cloning orexpression vector is not pure in that it may comprise only a smallpercentage of the material in the cell in which it resides. Such anucleic acid is isolated, however, as the term is used herein.

“Sequence homology” or “sequence identity” are used hereininterchangeably for nucleotides and amino acids sequences determinedusing FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res.,1997, 25, 3389, which is incorporated herein by reference in itsentirety) and PAUP* 4.ObIO software (D. L. Swofford, Sinauer Associates,Massachusetts). Briefly, the BLAST algorithm, which stands for BasicLocal Alignment Search Tool is suitable for determining sequencesimilarity (Altschul et al., J. Mol. Biol, 1990, 215, 403-410, which isincorporated herein by reference in its entirety). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide sequences would occur by chance.For example, a nucleic acid is considered similar to another if thesmallest sum probability in comparison of the test nucleic acid to theother nucleic acid is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001. “Percentage of similarity” or percentage of sequenceidentity” can be calculated using PAUP* 4.ObIO software (D. L. Swofford,Sinauer Associates, Massachusetts). The average similarity of theconsensus sequence is calculated compared to all sequences in thephylogenic tree.

The term “subject” is used throughout the specification to describe ananimal to whom treatment with the compositions according to the presentinvention is provided or administered. For treatment of those conditionswhich are specific for a specific subject, such as a human being or suchas a mammal, the term “patient” may be interchangeably used. In someinstances in the description of the present invention, the term“patient” will refer to human patients. In some embodiments, the subjectmay be a mammal to whom the present invention is provided oradministered. In some embodiments, the subject may be a domesticatedmammal to whom the present invention is provided or administered such asa horse, dog, cat, pig, cow, goat, sheep, llama, or other non-humananimal. In some embodiments, the subject is non-human. In someembodiments, the subject is a mammal suspected of having ahyperproliferative disorder. In some embodiments, the subject is ananimal diagnosed with malignant cancer and suspected of havingmetastatic cancer.

Isolated Nucleic Acid Sequences and Genetic Constructs

The invention provides for compositions comprising isolated nucleic acidmolecules and nucleic acid sequences disclosed herein. The inventionalso provides for kits comprising isolated nucleic acid molecules andnucleic acid sequences disclosed herein. The invention also provides forkits comprising isolated nucleic acid molecules and nucleic acidsequences disclosed herein.

In some embodiments, expressible forms of sequences that encode the oneor more polypeptides disclosed herein are found on the same nucleic acidmolecule that is transformed into one or more non-pathogenicmicroorganisms disclosed herein.

In some embodiments, expressible forms of sequences that encode the oneor more enzymes disclosed herein occur on a separate nucleic acidmolecule from the nucleic acid molecules that contain expressible formsof sequences that encode one or more toxin/antidote proteins. In. someembodiments, expressible forms of sequences that encode the one or moreenzymes and expressible forms of sequences that encode one or more ofthe toxin/antidote proteins occur on one nucleic acid molecule that isseparate from the nucleic acid molecule that contain expressible formsof sequences that encode one or more of the polarization proteinsdescribed herein. Multiple different nucleic acid molecules can beproduced and delivered according to the present invention andtransformed into the non-pathogenic bacterium described herein. Forexample, in some embodiments, expressible forms of sequences that encodeone or more enzymes described herein occur on separate nucleic acidmolecule from the nucleic acid molecules that contain expressible formsof sequences that encode one or more of the polarization proteins whichoccur on separate nucleic acid molecule from the nucleic acid moleculesthat contain expressible forms of sequences that encode thetoxin/antidote proteins. In such cases, all three molecules aredelivered to the individual.

The nucleic acid molecule(s) may be provided as plasmid DNA, the nucleicacid molecules of recombinant vectors or as part of a full or partialartificial bacterial chromosome. In some embodiments, the geneticconstruct or artificial bacterial chromosome is no more than about 50kilobases (kb) in nucleic acid base pair length. In some embodiments,the genetic construct or artificial bacterial chromosome is no more thanabout 45 kb in length. In some embodiments, the genetic construct orartificial bacterial chromosome is no more than about 40 kb in length.In some embodiments, the genetic construct or artificial bacterialchromosome is no more than about 35 kb in length. In some embodiments,the genetic construct or artificial bacterial chromosome is no more thanabout 30 kb in length. In some embodiments, the genetic construct orartificial bacterial chromosome is no more than about 25 kb in length.In some embodiments, the genetic construct or artificial bacterialchromosome is no more than about 20 kb in length. In some embodiments,the genetic construct or artificial bacterial chromosome is no more thanabout 15 kb in length. In some embodiments, the genetic construct orartificial bacterial chromosome is no more than about 10 kb in length.In some embodiments, the genetic construct or artificial bacterialchromosome is no more than about 5 kb in length. In some embodiments,the genetic construct or artificial bacterial chromosome is no more thanabout 2.5 kb in length.

Alternatively, in some embodiments, the nucleic acids that encode theone or more polypeptides and/or one or more proteins described hereinmay be transformed into any one or more non-pathogenic microorganismsdisclosed herein lie on only one plasmid. In some embodiments thenon-pathogenic microorganism or organisms do not comprise more than oneplasmid that encodes the one or more polypeptides and/or one or moreproteins described herein.

Genetic constructs may comprise a nucleotide sequence that encodes oneor more of the polypeptides or proteins described herein operably linkedto regulatory elements needed for gene expression. According to theinvention, combinations of gene constructs that include one thatcomprises an expressible form of the nucleotide sequence that encodes athe one or more polypeptides disclosed herein and one that includes anexpressible form of the nucleotide sequence that encodes are provided.Incorporation into a living cell of the DNA or RNA molecule(s) thatinclude the one or combination of genetic constructs results in theexpression of the DNA or RNA and production of the polypeptides orproteins described herein. Incorporation into one or more non-pathogenicmicroorganisms results in a composition comprising the genetic constructor isolated nucleic acid sequences disclosed herein.

In order to maximize protein production of any of the one or morepolypeptides disclosed herein, regulatory sequences may be selectedwhich are well suited for gene expression in the cells the construct isadministered into. Moreover, codons may be selected which are mostefficiently transcribed in the cell. One having ordinary skill in theart can produce DNA constructs that are functional in the cells.

The present invention provides for a genetic construct that comprises: afirst nucleic acid sequence encoding an enzyme, or functional fragmentthereof; a second nucleic acid sequence encoding a polarization protein,or functional fragment thereof; and a third nucleic acid sequenceencoding a combination of a toxin and an antidote. In some embodiments,the present invention relates to a composition comprising a geneticconstruct that comprises: a first nucleic acid sequence encoding anenzyme, or functional fragment thereof; a second nucleic acid sequenceencoding a polarization protein, or functional fragment thereof; and athird nucleic acid sequence encoding a combination of a toxin and anantidote. In some embodiments, the present invention relates to acomposition comprising a genetic construct that comprises: a firstnucleic acid sequence encoding an enzyme, or functional fragmentthereof; a second nucleic acid sequence encoding a polarization protein,or functional fragment thereof; and a third nucleic acid sequenceencoding a combination of a toxin and an antidote wherein the enzyme, orfunctional fragment thereof, catalyzes excitation of a portion of asubstrate or the release of a portion of a substrate when the substrateis present in the blood of a subject. In some embodiments, the presentinvention relates to a composition comprising a genetic construct thatcomprises: a first nucleic acid sequence encoding an enzyme, orfunctional fragment thereof; a second nucleic acid sequence encoding apolarization protein, or functional fragment thereof; and a thirdnucleic acid sequence encoding a combination of a toxin and an antidotewherein the enzyme, or functional fragment thereof, catalyzes excitationof a portion of a substrate or the release of a portion of a substratewhen the substrate is exposed to the enzyme or functional fragmentthereof. In some embodiments, the genetic construct is modified so that,upon introduction or transformation into a cellular host (such as one ormore of the non-pathogenic microorganisms disclosed herein, expressionof the one or more first, second, or third nucleic acids is sufficientto allow maximum protein expression of the nucleic acids withoutcompromising the metabolic pathways of the microorganism that allowgrowth of the microorganism in a certain microenvironment. Amodification of gene expression, also referred to herein as a modulationof gene expression, can be a disruption or inhibition of the naturalregulation of expression, an overexpression, an inhibition ofexpression, or a complete abolishment of expression of a given gene. Theinsertion of a heterologous promoter upstream of a native gene sequence,for example the consensus TATAAT or TTGACA sequences, or the deletion ofregulatory sequences within a promoter, for example regulatory sequencesthat mediate the inhibition of the consensus TATAAT or TTGACA sequences,are examples of a disruption or inhibition of the natural regulation ofexpression. Strategies for the modulation of gene expression may includegenetic alterations, for example by recombinant technologies, such asgene targeting or viral transductions, or non-genetic alterations, forexample environmental alterations known to result in the up- ordown-regulation of gene expression, or transient delivery of modulators,for example drugs or small RNA molecules to the target cells. Methodsfor genetic and non-genetic alterations of microbes are well known tothose of skill in the art, and are described, for example, in J.Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001); David C.Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual, Cold SpringHarbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon,Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics andMolecular Biology, Part A, Volume 194 (Methods in Enzymology Series,194), Academic Press (Mar. 11, 2004); Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B,Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition(Jul. 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to YeastGenetics and Molecular and Cell Biology, Part C, Volume 351, AcademicPress; 1st edition (Jul. 9, 2002); Gregory N. Stephanopoulos, Aristos A.Aristidou and Jens Nielsen, Metabolic Engineering: Principles andMethodologies, Academic Press; 1 edition (Oct. 16, 1998); and ChristinaSmolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRCPress; 1 edition (Jul. 28, 2009), all of which are incorporated byreference herein in their entireties.

The present invention also related to isolated expression vectors orgenetic constructs that encode one or more of the polypeptides orfunctional fragment disclosed herein or compositions comprising any oneor combination of the nucleic acid sequences disclosed herein. In someembodiments, the expression vector includes a coding nucleic acid, forexample, a nucleic acid encoding an enzyme or functional fragment, apolarization protein or functional fragment thereof and/or atoxin/antidote combination gene product, operably linked to aconstitutive promoter. The term “constitutive promoter” refers to apromoter that allows for continual transcription of its associated gene.In some embodiments, the expression vector includes a coding nucleicacid, for example, a nucleic acid encoding a an enzyme or functionalfragment, a polarization protein or functional fragment thereof and/or atoxin/antidote combination gene product, operably linked to an induciblepromoter. The term “inducible promoter”, interchangeably used hereinwith the term “conditional promoter”, refers to a promoter that allowsfor transcription of its associated gene only in the presence or absenceof biotic or abiotic factors. Drug-inducible promoters, for exampletetracycline/doxycycline inducible promoters, tamoxifen-induciblepromoters, as well as promoters that depend on a recombination event inorder to be active, for example the cre-mediated recombination of loxPsites, are examples of inducible promoters that are well known in theart.

In some embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 80% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 85% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 90% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 91% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 92% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 93% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 94% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 95% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 96% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 97% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 98% sequence identity to SEQ ID NO:1. Insome embodiments, the nucleic acid or genetic construct comprises anucleotide sequence at least 99% sequence identity to SEQ ID NO:1. Insome embodiments, the compositions disclosed herein comprise a nucleicacid or genetic construct consisting of SEQ ID NO:1.

In some embodiments, the non-pathogenic microorganism comprises any oneor more of any of the nucleic acid sequence disclosed herein or anyfunctional fragment thereof with any sequence homology disclosed herein(at least 70% through at least 99% sequence homology).

Non-Pathogenic Organisms

In some embodiments, the invention relates to a composition comprising anon-pathogenic microorganism that comprises a first nucleic acidsequence encoding an enzyme or functional fragment thereof, a secondnucleic acid sequence encoding one or more polarization polypeptides orfunctional fragments thereof, a third nucleic acid sequence encoding abacterial toxin or functional fragment thereof, and a fourth nucleicacid sequence encoding an antidote to the bacterial toxin. In order tomanufacture or produce such a non-pathogenic microorganism, one ofordinary skill in the art would contact the microorganism with acomposition comprising each of the first, second, third, or fourthnucleic acid sequences. Methods to deliver expression vectors orexpression constructs into microbes, for example, into non-pathogenicmicroorganisms, are well known to those of skill in the art. Nucleicacids, including expression vectors, can be delivered to prokaryoticmicrobes by various methods well known to those of skill in the relevantbiological arts. Methods for the delivery of nucleic acids to a microbein accordance to some aspects of this invention, include, but are notlimited to, different chemical, electrochemical and biologicalapproaches, for example, heat shock transformation, electroporation,transfection, for example liposome-mediated transfection,DEAE-Dextran-mediated transfection or calcium phosphate transfection. Insome embodiments, a nucleic acid construct, for example an expressionconstruct comprising any combination of the first, second, third, and/orfourth nucleic acid sequences disclosed herein, is introduced into thehost microbe using a vehicle, or vector, for transferring geneticmaterial. Vectors for transferring genetic material to microbes are wellknown to those of skill in the art and include, for example, plasmids,artificial chromosomes, and viral vectors. Methods for the constructionof nucleic acid constructs, including expression constructs comprisingconstitutive or inducible heterologous promoters, knockout and knockdownconstructs, as well as methods and vectors for the delivery of a nucleicacid or nucleic acid construct to a microbe are well known to those ofskill in the art, and are described, for example, in J. Sambrook and D.Russell, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press; 3rd edition (Jan. 15, 2001); David C. Amberg, DanielJ. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A ColdSpring Harbor Laboratory Course Manual, Cold Spring Harbor LaboratoryPress (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie,and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, PartA, Volume 194 (Methods in Enzymology Series, 194), Academic Press (Mar.11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Geneticsand Molecular and Cell Biology, Part B, Volume 350 (Methods inEnzymology, Vol 350), Academic Press; 1st edition (Jul. 2, 2002);Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen,Metabolic Engineering: Principles and Methodologies, Academic Press; 1edition (Oct. 16, 1998); and Christina Smolke, The Metabolic PathwayEngineering Handbook: Fundamentals, CRC Press; 1 edition (Jul. 28,2009), all of which are incorporated by reference herein in theirentireties.

Some aspects of this invention relate to cultures of geneticallymodified microbes provided herein. In some embodiments, the culturecomprises a genetically modified microbe provided herein and a medium,for example, a liquid medium. In some embodiments, the culture comprisesa genetically modified microbe provided herein and a carbon source, forexample, a carbohydrate source, or an organic acid or salt thereof. Insome embodiments, the culture comprises a genetically modified microbeprovided herein and a salt and/or buffer establishing conditions ofsalinity, osmolarity, and pH, that are amenable to survival, growth,and/or catalytic activity of the encoded enzyme. In some embodiments,the culture comprises an additional component, for example, an additive.Non-limiting examples of additives are nutrients, amino acids, albumin,growth factors, enzyme inhibitors (for example protease inhibitors),fatty acids, lipids, hormones (e.g., dexamethasone and gibberellicacid), trace elements, inorganic compounds (e.g., reducing agents, suchas manganese), redox-regulators (e.g., antioxidants), stabilizing agents(e.g., dimethylsulfoxide), polyethylene glycol, polyvinylpyrrolidone(PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g., NaCl),chelating agents (e.g., EDTA, EGTA), and enzymes (e.g., cellulase,dispase, hyaluronidase, or DNase). In some embodiments, the culture maycomprise a drug inducing or inhibiting transcription from a conditionalor inducible promoter, for example doxicycline, tetracycline, tamoxifen,IPTG, hormones, or metal ions. While the specific culture conditions,for example, the concentration of the carbon source, will depend uponthe respective engineered microorganism to be cultured, general methodsand culture conditions for the generation of microbial cultures are wellknown to those of skill in the art, and are described, for example, inJ. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001); David C.Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual, Cold SpringHarbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon,Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics andMolecular Biology, Part A, Volume 194 (Methods in Enzymology Series,194), Academic Press (Mar. 11, 2004), all of which are incorporated byreference herein.

In some embodiments, the genetically modified non-pathogenic microbe ormicroorganism exhibits a growth advantage over wild type microbes of thesame kind and/or over other microbes, for example, microbes commonlyfound to contaminate microbial cultures for scaling up production ofcultures to large volumes. In some embodiments, the growth and/orproliferation advantage of an engineered microbe provided by aspects ofthis invention translates into the possibility of using non-sterileculturing and fermentation conditions for production, because theproblem of culture overgrowth by contaminating microbes is mitigated orcompletely abolished. In some embodiments, an engineered microbeprovided by aspects of this invention is cultured under non-sterileconditions. For example, in some embodiments, non-sterilized feedstock,non-sterilized culture media, non-sterilized supplements, or anon-sterilized bioreactor (e.g. an open reactor under non-sterileconditions) is used for microorganism division.

A variety of different microbes can be genetically modified according tosome aspects of this invention and used for scale-up and/or isolationfor eventual animal ingestion, for example, various strains ofnon-pathogenic E. coli. In some embodiments, the invention provides fora composition comprising one or a combination of non-pathogenicmicroorganisms chosen from: probiotic bacteria is chosen fromEscherichia spp., Firmicutes spp., Bacteroidetes spp., Lactobacillusspp., Bifidobacteria spp., or Acidopholus spp. In some embodiments, theprobiotic is selected from Lactobacillus, Bifidobacteria, andAcidopholus. In some embodiments, the bacteria is harvested from a humanor animal sample and transformed as described herein. with our system.The samples may be from stool samples (probiotics) or human/mouse tumorsamples (bacteria that have potential to be very tumor selective). Insome embodiments, the non-pathogenic microorganism is E. coli Nissle1917 (EcN). In some embodiments, the non-pathogenic microorganism is astrain of E. coli but not the Nissle 1917 (EcN).

The present invention relates to compositions comprising any one orcombination of non-pathogenic microorganisms disclosed herein wherein atleast one of the microorganisms comprises the first, second, third,and/or fourth nucleic acid sequences disclosed herein either alone or incombination. In some embodiments, the invention relates to compositionscomprising any one or combination of non-pathogenic microorganismsdisclosed herein wherein at least one of the microorganisms comprises anucleotide sequence encoding a mutated virulence factor. For instance,in some embodiments, the non-pathogenic microorganism comprises amutation in its Type I, II, III, IV, V, or VI secretion system whichdoes not allow transport of host toxins and/or host immunogenic proteinsout of the cell.

In some embodiments, fermentation processes for large-scale microbe celldivision may be carried out in bioreactors, isolated and thenresuspended in an amount or dosage form disclosed herein. As usedherein, the terms “bioreactor” and “fermentor”, which areinterchangeably used, refer to an enclosure, or partial enclosure, inwhich a biological and/or chemical reaction takes place, at least partof which involves a living organism or part of a living organism. A“large-scale bioreactor” or “industrial-scale bioreactor” is abioreactor that is used to generate large volumes of non-pathogenicmicroorganisms for large scale isolation. Large scale bioreactorstypically have volumes in the range of liters, hundreds of liters,thousands of liters, or more.

Other examples of bacteria that can be modified for use in the inventioninclude food-grade bacterial strains. In some embodiments, thenon-pathogenic bacteria is chosen from the bacterials strains identifiedin Humarán et al., Microbial Cell Factories 2011, 10(Suppl 1):S4;Shrivastava et al., PlosOne, August 2008, Volume 3, Issue 8: e2955; andT. Danino et al., ACS Synth. Biol. 2012, 1, 465-470, each of which areincorporated by reference in their entireties.

The invention relates to compositions comprising any of thenon-pathogenic microorganisms disclosed herein, wherein the compositionsare in a solid or liquid dosage form an wherein the microorganisms arein an effective amount to colonize a tumor. The invention relates tocompositions comprising any of the non-pathogenic microorganismsdisclosed herein, wherein the compositions are in a solid or liquiddosage form an wherein the microorganisms are in an effective amount tocolonize a tumor and adequate produce enzyme or substrate in quantitiesdetectable in urine. The invention relates to compositions comprisingany of the non-pathogenic microorganisms disclosed herein, wherein thecompositions are in a solid or liquid dosage form an wherein themicroorganisms are in an effective amount to colonize a tumor andadequate produce enzyme or substrate in quantities detectable in urineby visual inspection. The invention relates to compositions comprisingany of the non-pathogenic microorganisms disclosed herein wherein thecompositions are in a solid or liquid dosage form. In addition to thecompositions disclosed in either solid or liquid form, the formulationsand compositions of the present invention may also contain optionalingredients to enhance the characteristics of the solid dosage form,maintain the integrity of microorganisms (in dried, lyophilized, dormantor sporulated forms) during the formulation process, and/or enhance thesafety of the formulation. Any additional components may be compatiblewith the other ingredients in the formulations of the invention, inparticular the active ingredients, and may be inert. If inert, theadditional component does not adversely affect the osmolarity,osmolality, or isotonicity of the formulations or interfere, to ameasurable degree, with the biological function of the non-pathogenicmicroorganism. Additional optional ingredients that may be used in theformulations of the invention include, for example, coatings, diluents,binders, glidants, lubricants, colors, disintegrants, flavors,sweeteners, polymers or waxes.

Non-limiting examples of diluents include various types of starch,cellulose, crystalline cellulose, microcrystalline cellulose, lactose,fructose, sucrose, mannitol or other sugar alcohols, kaolin, calciumphosphate or sulfate, inorganic salts such as sodium chloride andpowdered sugar. Powdered cellulose derivatives are also useful. In someembodiments of the invention, the formulation does not include adiluent.

Lubricants may be included in the formulations of the invention. Suchlubricants include, but are not limited to, magnesium stearate,potassium stearate, talc, stearic acid, sodium lauryl sulphate, andparaffin. In some embodiments of the invention, the colonic purgativeformulation further comprises magnesium stearate. Lubricants serve tofacilitate the manufacturing of a solid dosage form. In some embodimentsof the invention, the formulation does not comprise a lubricant.

Additional suitable ingredients also include, but are not limited to,carriers, such as sodium citrate and dicalcium phosphate; fillers orextenders, such as stearates, silicas, gypsum, starches, lactose,sucrose, glucose, mannitol, talc, and silicic acid; binders, such ashydroxypropyl methylcellulose, hydroxymethyl-cellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose, and acacia; humectants, such asglycerol; disintegrating agents, such as agar, calcium carbonate, potatoand tapioca starch, alginic acid, certain silicates, colloidal silicondioxide, sodium starch glycolate, crospovidone, and sodium carbonate;solution retarding agents, such as paraffin; absorption accelerators,such as quaternary ammonium compounds; wetting agents, such as cetylalcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; stabilizers, such as fumaric acid; coloring agents;buffering agents; dispersing agents; preservatives; organic acids; andorganic bases.

In some embodiments of the instant invention, the tablet or capsules mayalso include inert dispersal agents which will facilitate dissolution ofthe a solid dosage form of the non-pathogenic microorganisms in thestomach of the patient. Preferably, the dispersal agent is apharmaceutically acceptable dispersant and is one which also produces noappreciable osmotic effects. Examples of acceptable dispersants includemicrocrystalline cellulose (which is also useful as a compacting agent)and anhydrous lactose. In some embodiments, the dispersal agent isAC-DI-SOL, a cross-linked starch.

In some embodiments of the present invention, the formulation orcomposition may also include a buffering agent to minimize any acidimbalance which may accompany ingestion of the compositions disclosedherein. Suitable buffering agents include magnesium hydroxide, aluminumhydroxide, calcium carbonate and magnesium carbonate. In someembodiments, the formulation does not include a buffering agent.

In some embodiments of the invention, an additional component in theformulations of the invention may function to maintain the electrolytebalance in a subject after ingestion of a liquid dosage form of thecompositions disclosed herein. For example, formulations of theinvention may further comprise calcium, phosphate, potassium, magnesium,other anions, or salts thereof.

The present invention further relates to a food product comprising anyone of the disclosed non-pathogenic organisms disclosed herein anoptionally any one of combination of the diluents, buffering agents,dispersal agents, carriers, lubricants disclosed herein. In someembodiments, the food product is selected from the group consisting of:beef products, poultry products, pork products, and dairy products;wherein the food product comprises any one or combination ofmicroorganisms disclosed herein. In some embodiments, the food productis a dairy product. If a dairy product one can create the startingmaterial for a fermentation method where the dairy product comprisescombination or mixture of an aqueous protein source and a fat source.The dairy product can be or comprise a milk concentrate, a milksubstrate, a whey concentrate, a whey substrate, cheese curd, and thelike, or combinations of these dairy substances with each other or incombination with a supplemental protein or fat source. The dairy productgenerally will be in the form of an aqueous protein and fat sourcecombination. It also can be in emulsion form. The same or differentdairy product compositions can be used as the starting material used inpreparing one or more flavor components.

The dairy products useful as the starting material generally have totalsolids contents of about 10 to about 50 percent, protein contents ofabout 10 to about 19 percent, fat contents of about 5 to about 30percent, and lactose contents of about 0.1 to about 10 percent.Preferably, they have total solids contents of about 25 to about 47percent, protein contents of about 12 to about 17 percent, fat contentsof about 18 to about 25 percent, and lactose contents of about 0.5 toabout 5 percent. The moisture levels of the dairy product are generallyfrom about 50 to about 90 percent, preferably from about 53 to about 75percent.

The protein source can be a dried protein or concentrated material andis a dairy ingredient, such as milk protein concentrate, fractionatedmilk protein, concentrated milk fat, whey protein concentrate, driedwhey, non-fat dry milk, milk protein isolate, whey protein isolate, ormixtures thereof. Other protein sources, such as soy protein, cornprotein, wheat protein, and/or rice protein can be used in part or asthe sole protein source. The fat source is preferably a milk fat such asanhydrous milk fat, butter, cream, or mixtures thereof. Other non-dairyfat sources, such as vegetable oil, can be used in part or as the solefat source. The pH of the dairy concentrate or substrate is generally inthe range of about 6 to about 7 and preferably in the range of about 6.5to about 6.7. In general, at least one of the protein and fat sourceswill include a dairy ingredient in the practice of this invention toprovide a highly useful starting material from which various flavorswhich are normally or otherwise associated with food products can bedeveloped.

A dried protein source, if used, is reconstituted with water. The wateris used at a level sufficient to provide total moisture of from about 50to about 90 percent, preferably from about 53 to about 75 percent in thesubstrate. The reconstituted protein source is combined with the fatsource to provide the substrate. If necessary, the pH of the substratecan be lowered to the proper range (i.e., about 4.6 to about 6 andpreferably about 4.8 to about 5.6) by the addition of an edible acid orby use of a lactic acid producing microorganism. Suitable edible acidsare non-toxic, inorganic or organic acids, which include hydrochloricacid, acetic acid, maleic acid, tartaric acid, citric acid, phosphoricacid, lactic acid, and mixtures thereof. In preparing the milkconcentrate, a homogenization device can be used to reduce the fatdroplet particle size and insure homogeneity of the substrate.

In some embodiments, the dairy product used as the starting material isan aqueous milk-derived concentrate or substrate that is a fluid milkconcentrate prepared by ultrafiltration (alone or even more preferablycombined with diafiltration) or a reconstituted milk substrate preparedfrom a mixture of an ultrafiltered (UF) or ultrafiltered/diafiltered(UF/DF) milk powder and milk fat. The starting material can be an UF/DFmilk having the characteristics such as described in U.S. Pat. No.6,406,724. These milk concentrates can be used as is or in combinationwith a supplemental fat source to provide the starting material.

Dairy products useful as starting materials for the methods of thepresent invention can be prepared from concentrated whole or skim milkwith, if desired, added cream or anhydrous milk fat (AMF). The cream orAMF generally is added in an amount of about 0 to about 20 percent,preferably about 2 to about 15 percent, by weight of the mixture. In oneembodiment for making the dairy product, skim milk is subjected toconventional ultrafiltration/diafiltration techniques to produce anabout 3.times. to about 8.times. (preferably about 5.times.) milkconcentrate product. Cream or anhydrous milk fat or a combinationthereof is mixed with the milk concentrate. In one exemplarynon-limiting embodiment, the resulting mixture is homogenized, andpasteurized under high temperature short time (HTST) conditions, such asat about 76.degree. C. for about 16 seconds in a heat exchanger, andthen it is cooled to about 21 to about 27.degree. C. The resulting dairyproduct can be used as the starting material that is subjected tofermentation to prepare the specific flavoring components of the presentinvention. In some embodiments, about 1 to about 2 percent salt is addedto the dairy product prior to treatment with the variousenzymes/cultures/additives to produce the specific flavoring components.The pasteurized dairy product is a relatively viscous liquid, preferablycontaining about 25 to about 47 percent solids.

Dairy product comprising fluid milk concentrate or whey concentrate, AMFor the like and preferably containing about 1 to about 2 percent salt,can then be divided into one, two or three portions, each of which istreated (i.e., fermented) with specific batches of non-pathogenicmicro-organisms of the present invention and/or substrates specific forthe enzymes of the present invention. In some embodiments, the presentinvention optionally comprises cultures, adjuncts, and other additivesfor predetermined time periods sufficient to develop specific flavorcharacteristics. Specific enzymes, cultures, diluents, substrates (suchas IPTG), adjuncts, and other additives are provided herein.

In some embodiments, the food product comprises any one or combinationof non-pathogenic microorganisms disclosed herein comprising any one orcombination of any nucleic acid sequence disclosed herein.

Enzymes and Substrates

The invention provides a composition comprising a nucleic acid sequencethat encodes one or more enzymes capable of catalyzing one or moreconcurrencies of reactions that excite or release a substrate, orportion of a substrate, or a reaction product detectable in the blood,urine, and/or feces of a subject after administration to the subject. Insome embodiments, one or more enzymes capable of catalyzing one or moreconcurrencies of reactions that excite or release a substrate, orportion of a substrate, or a reaction product emits a visible wavelengthof light under white light. In some embodiments, upon administration ofthe nucleic acid to an animal subject, the nucleic acid sequence encodesan enzyme capable of modifying or cleaving substrate into a coloredproduct, wherein the colored product is excreted by a subject in urineif the cleaving or modifying takes place in a subject. In someembodiments, upon administration of the nucleic acid to an animalsubject, the nucleic acid sequence encodes an enzyme capable ofmodifying or cleaving substrate into a colored product excreted by asubject in urine. In some embodiments, upon administration of thenucleic acid to an animal subject, the nucleic acid sequence encodes anenzyme capable of modifying or cleaving substrate into a productexcreted by a subject in urine, wherein the product is excitable orfluorescent under conditions sufficient to excite the product and toemit a wavelength of light visible to the human eye. In someembodiments, the product is excitable or fluorescent under white light,UV light, or infrared light. In some embodiments, the substrate, portionthereof, or product of enzyme catalysis is excitable or fluorescentunder white light but is not excitable under UV light, or infraredlight. In some embodiments, the substrate, portion thereof, or productof enzyme catalysis is excitable or fluorescent under UV light orinfrared light, but not any visible wavelength of light. In someembodiments, the substrate, portion thereof, or product of enzymecatalysis the product is excitable or fluorescent under white light, UVlight, or infrared light, but emits a wavelength of light detectable byvisual inspection of the human eye.

In some embodiments, the enzyme may be chosen from: glucoronidase orhuman lactase. If such enzymes are used, in some embodiments, additionaladministration of effective amounts of their respective known substratesto a subject should be used to detect the presence, absence, orquantities of the substrate or portion thereof or a reaction product inthe urine of the subject. In some embodiments, amounts of glucoronidaseor human lactase or portions thereof may be measured in the urine of thesubject.

lacZ component systems as described in Jiayuan Quan et al., NatureBiotechnology 29, 449-452 and Mol Pharm. 2010 Feb. 1; 7(1): 60-74. mayalso be used in some embodiments for detection of components of enzymesrather than detection of substrate or products of enzymatic reactions,such references are herein incorporated by reference in its entirety.

In some embodiments, different strains of microorganisms that comprisethe plasmid stability machinery disclosed herein comprise genes thatencode pigments that can be secreted and act as detectable agent in theurine or body of a subject. One such pigment is violacein.

In some embodiments, the non-pathogenic microorganism comprises

Polarization Sequences

In some embodiments, the second nucleic acid sequence encodes apolarization protein, or functional fragment thereof, wherein thepolarization protein facilitates the inclusion of the first, second, andthird nucleic acid sequences in the non-pathogenic microorganism and itsprogeny upon cellular division. In some embodiments, the polarizationprotein is a cytoplasmic microtubule associated protein or a combinationof two cytoplasmic microtubule associated proteins. In some embodiments,the polarization protein is AlpA (see, Example 5 for sequence).Polarization protein systems and their use are well known in the art. Aphylogenetic search was conducted and uncovered more than 35 highlydivergent families of actin-like proteins (Alps) in bacteria. Theirgenes are found primarily on phage genomes, on plasmids and onintegrating conjugative elements, and are likely to be involved in avariety of functions. Three Alps were characterized and all formfilaments in the cell. The filaments of Alp7A, a plasmid partitioningprotein and one of the most divergent of the Alps, display dynamicinstability and also treadmill. Alp7A requires other elements from theplasmid to assemble into dynamic polymers in the cell. Most if not allof the Alps are indeed actin relatives, and that actin is very wellrepresented in bacteria.

Alp7A is a plasmid partitioning protein, as are the previouslycharacterized ParM and AlfA amino acid sequences. In some embodiments,Alp7A is fued with a fluorescent protein, such as GFP. IN someembodiments, Alp7A-GFP fusion protein retained its function and could beused interchangeably with Alp7A itself. In some embodiments, the methodsdisclosed herein can correlate Alp7A function with its behaviour in thecell and assess whether the actin properties of Alp7A were required forits function. We found that mutations in two amino acids that would bepredicted to interact with nucleotide on the basis of actin biochemistryand structural biology disrupted Alp7A polymerization dynamics. TheD212A mutation, which abolished filament formation, wasindistinguishable from a null mutation in a plasmid stability assay. TheE180A mutation, which permitted filaments to form but eliminated theirdynamic properties, was almost as crippling. The analogous mutation inParM (E148A) eliminates ATP hydrolysis, and leads to stable filamentformation. It follows that in order to function as a plasmidpartitioning protein, Alp7A must behave as a bacterial actin: it mustpolymerize into filaments with dynamic properties.

In some embodiments, the plasmids, compositions, pharmaceuticalcompositions of the claimed invention comprise a nucleic acid sequencecomprising SEQ ID NO: 5 or a nucleic acid sequence comprising SEQ ID NO:6 and SEQ ID NO:7.

In some embodiments, the plasmids, compositions, pharmaceuticalcompositions of the claimed invention comprise a nucleic acid sequencecomprising parMRC. parM has similar extra elements like alp7R, for thatsystem it is parM (actin)+parRC (binding elements)=parMRC. Each of thesesystems has 1 actin-like element (alp7A in our case) and 1 or morebinding element (alp7R in our case) that the actin piece connects to, sothat the plasmids can push each other apart. In some embodiments, thepolarization protein comprises two amino acid elements, a first aminoacid actin-like element, and a second binding element. In someembodiments, the two amino acid elements are alp7A and alp7R orfunctional fragments thereof. In some embodiments, the two amino acidelements are parM and parRC or functional fragments thereof, whereinparR is:

(SEQ ID NO: 15) ATGGCTAAAAACCCTATCTCAAATAAGGCAGATAACGACCGGATTCAGATCCGGTCTTTCTGGATATCCGAAAGAAAAGCACCCTATGTTTATAGTTTCTTGAAAAAAACAGAACTTTCTCATAGGGGTGACCAACTGGATTTAATTAGGTCGGCTATTAGTACCGGGTTGGTATTGAATAATTTATTTCCTGACTTGGCAAATTTTATAAATGGTTTAAACGAAAGATTAACACTTGCAGATCTTAATAGGTTTCTGAATGATGGAAATACTATAGATACTGAACCTAAGCCTCCTATTAATGTATTGCTAGAGAATGTCTTAGATCAAAAGTTTAAGGAGTATTTAACACCTCTACAATTAGATAATTCAAAGCAAGATTCTGTTTCTGTAAAAGAAACCTTCCTTGTACAAAAGGAACATGCCTGCTTTGGTGTGAAGATTGAAAATGAGGGAAGCGATACCTCTATACCATCTGAAAGCCCACTTTCTTCAGATGCATCCAAAATTTCAAAAGAAAAGTCCATTTCCGCTGTGGTGCCAGTGCTAGAAAAAGTATCGGATGAAAATCAAACCGCCTCCATAAGCATAAAATCTAAAGCTAAGGCAAACAAGCGACTGGCAACTTTGGCAAGAT AGor a functional fragment thereof; and Wherein parC is (SEQ ID NO: 16)TCATGCGTGGCCCCATTGCTGATGATCGGGGTACGCCAGGTGCAGCACTGCATCGAAATTGGCCTTGCAGTAGCCGTCCAGCGCCACCCGCGAGCCGAACGCCGGCGAAAGGTACTCGACCAGGCCGGGCCGGTCGCGGACCTCGCGCCCCAGGACGTGGATGCGCCGGCCGCGTGTGCCGTCGGGTCCAGGCACGAAGGCCAGCGCCTCGATGTTGAAGTCGATGGATAGAAGTTGTCGGTAGTGCTTGGCCGCCCTCATCGCGTCCCCCTTGGTCAAATTGGGTATACCCATor a functional fragment thereof. Wherein parM is (SEQ ID NO: 18)ATGTTGCTATTCATTGATGACCGTTCAACAAACATCAAACTACAGTCGCAGGAAAGCGACCGAACAATTAAACAGCACATTAGCCCGAACAGCTTCAAACGCGAGTCGCCAGTCTCTTTTGGTGATAAAAAGGTCTTTAACTAGAGACTGAACGGCGAACAGTATTGATTTGATCCAATCAGCCCGGATGCTGTAGTCACAACCAATATCGCATGGCAATACAGCGACGTTAATGTCGTTGCAGTGCATCACGCCTTACTGACCAGTGGTCTGCCGGTAAGCGAAGTGGATATTGTTTGCACACTTCCTCTGACAGAGTATTACGACAGAAATAACCAACCCAATACGGAAAATATTGAGCGTAAGAAAGCAAACTTCCGGAAAAAAATTACATTAAATCGCGGGGATACATTCACAATAAAAGATGTAAAAGTCATGCCTGAATCTATACCGGCAGGTTATGAAGTTCTACAAGAACTGGATGAGTTAGATTCTTTATTAATTATAGATCTCGGGGGCACCACATTAGATATTTCTCAGGTAATCGGGAAATTATCGGGGATCAGTAAAATATACGGAGACTCATCTCTTGGTGTCTCTCTGGTTACATCTGCAGTAAAAGATGCCCTTTCTCTTGCGAGAACAAAAGGAAGTAGCTATCTTGCTGACGATATAATCATTCACAGAAAAGATAATAACTATCTGAAGCAACGAATTAATGATGAGAACAAAATATCAATAGTCACCGAAGCAATGAATGAAGCACTTCGTAAACTTGAGCAACGTGTATTAAATACGCTCAATGAATTTTCTGGTTATACTCATGTTATGGTTATAGGCGGTGGCGCAGAATTAATATGCGATGCAGTAAAAAAACACACACAGATTCGTGATGAACGTTTTTTCAAAACCAATAACTCTCAATATGATTTAGTTAACGGTATGTATCTCATAGGTAATTAA or a functional fragment thereof.

In some embodiments, the two amino acid elements are alfA and alfB orfunctional fragments thereof, wherein alfA and alfB are disclosed inNC_021809 incorporated by reference in its entirety.

alfA

(SEQ ID NO: 17) TTGACACTAACTACTGTAATTGATATCGGGAATTTTAGTACGAAGTACGCTTATAAGGACAAAAAACAAATTAAGGTCGGCAGTTTCCCTTCTATTCTCCATAGCTACAAGCCTTTAGAAGATTACGAGGGAATGGAAAGAGTAGAGTACAACGGCCTTGATTATTATGTTGGAGAAACCGTTAAGAACTTCTATTTCGGCCGTGAAGAACAAATGTATTTCGGCAATACAAGAAAAGGCCATATGGAAGGTCAAATTCGATTAGTATATGCTCTCTATACAATCTTTAAAGAGACTGGAAAGAAAGAATTTAACTTAATTCTAACTTGCCCATATGAAAGTATGGTTACAGATAAAAAATATTTCGTTCAACATTTTGAAGGAGAAAGAGAAGTTATCGTTGAAGGAAAGTCATTCAAATTCACTGTACATAATATCGTGATGGCTGCAGAGGGATTAGGAGCCCTAAACTTCTCAGATTCATTAAACTGCGTCATTGTAGATGCTGGTTCTAAGACATTAAACGTCCTTTATTTAATCAATGGGTCTATAAGTAAAATGGATAGCCATACTATTAATGGTGGGACGATCGACAATTCAATAATGGATTTGGCGAAGACTTTTGCTAAGACTTGCAGCAATATCGATTATGACTACCCTATTGTTTGTACAGGTGGTAAAGCAGAAGAAATGAAAGAATGCTTAGAGAATGTTGGATATTCCACTGTAAGTTCTGCCGAACTGGGTGAGGATAAACCATCTTACTATGTTAATTCAGTTGGATTGCTTCTAAAATACGGTAGGAAGTTTGAGGAGATGTTTGCGTGAToxin/Antidote Sequences

The present invention relates to a non-pathogenic microorganism thatcomprises a nucleic acid sequence that encodes a bacterial toxin andfurther comprises a nucleic acid sequence that encodes an antidote tothe bacterial toxin. In some embodiments, the nucleic acid sequenceencoding the toxin is on a first nucleic acid molecule and the nucleicacid sequence encoding the antidote to the toxin is on a second nucleicacid molecule. In some embodiments, the nucleic acid sequence encodingthe toxin and the nucleic acid sequence encoding the antidote to thetoxin are in a single plasmid.

Toxin and antidote combinations are known in the art. In someembodiments, the toxin and antidote combination is any of thetoxin/antidote combinations disclosed in Gerdes, et al., MolecularMicrobiology (1990) 4(11), 1807-1818, which is herein incorporated byreference in its entirety. The hok/sok system consists of two genes: hokand sok. The Hok protein is a potent cell-killing agent of 52 aminoacids (Gerdes et at. 1986b). The sok product is trans-acting antisenseRNA that represses hok gene expression at a post-transcriptional level(Gerdes et al. 1988). Since the initial cloning of the hok/sok locus, afamily of hok-homologous killer genes has been identified. The E.Colichromosome carries two hok homologues: re/Fat 34 minutes (Gerdes etal. 1986b), and getA at 0 minutes (Poulsen et al. 1989). Furthermore,Poulsen et al. showed that a wide range of distantly relatedGram-negative species also encoded two hok-homologous loci. The Fplasmid also carries two hok-homologous loci: srnB near the RepFIGregion (Akimoto et al. 1986; Saadi et al. 1987), and film in thetransfer leading region of F (Golub and Low, 1986). Interestingly, allof the hok-homologous killer genes mentioned here are regulated at apost-transcriptional level.

The parB locus of plasmid R1, comprising at most 580 base pairs of DNA,mediates efficient plasmid stabilization via postsegregational killingof plasmid free cells. The locus encodes two small genes, hok andsok.The hok gene product is a potent cell killing protein, the expression ofwhich is regulated by the sok product, an anti-sense RNA complementaryto the hok mRNA. The hok mRNA is extraordinarily stable, while thesok-RNA is rapidly degraded. The mechanism of postsegregational killingis explained by the differential decay of the hok and sok-RNA's: Innewborn plasmid free cells the prolonged persistence of the hok mRNAleads to synthesis of the Hok protein, thus ensuring a rapid andselective killing of these cells. As predicted from this simple model,any unstably inherited plasmid should become stabilized by carrying theparB locus. This important prediction was confirmed by testing a varietyof different replicons. Even plasmids replicating in as distantlyrelated organisms as Eschericia coli, Serratia marsescense, andPseudomonas putida were efficiently stabilized. The parB locus thereforeconstitutes a convenient and efficient plasmid stabilization cassette,useful in many gram negative species.

Plasmid instability is a primary impediment to industrial utilization ofrecombinant microorganisms. As the vector becomes more effective indirecting protein production, it becomes an increasing drain on cellularmetabolism. Betenbaugh et al. (1989) have shown that the growth rate ofthe plasmid-bearing cell is reduced relative to the plasmid-free cell ascloned-gene expression is increased through either plasmid amplificationor promoter induction. Hence, faster-growing, plasmid-free segregantscan rapidly outnumber the plasmid-bearing population and greatly reducethe yield of recombinant protein from the culture. In addition,structural instability through homologous recombination events can leadto plasmid derivatives that no longer produce the desired protein.

As a result of the segregational and structural instability, the plasmidinitially chosen for our studies, pMJR1750 (Stark, 1987), was extremelyunstable in the host strain AMA1004 (Casadaban et al., 1983), even inthe presence of antibiotics. This plasmid was chosen to help discern themetabolic impact of recombinant protein expression because it allowstranscription of-galactosidase to be tightly regulated by the additionof IPTG to the fermentation medium. In order to increase the stabilityof this vector, three changes were made to the host/plasmid system: therecA gene was deleted from the host, the antibiotic selection marker waschanged from ampicillin (Ap) to kanamycin (Kan), and the parB stabilitylocus of plasmid R1 (Gerdes, 1988) was added to the plasmid.

Structural instability should be reduced considerably by the recAdeletion. This deletion should severely limit homologous recombinationbetween the plasmid and chromosome since Csonka and Clark (1979) haveshown the Δ(srl-recA)306 mutation used in this work decreases the rateof recombination of the host during conjugation by a factor of 36,000.Additionally, Laban and Cohen (1981) have shown that arecA pointmutation lowers the frequency of recombination events within a plasmidby 100-fold.

To provide more effective selection pressure, the selectable marker waschanged from β-lactamase (Ap_(r)) to aminoglycoside3′-phosphotransferase II (APH, Kan_(r)). Unlike APH, β-lactamase istransported to the periplasmic space and leaks in sufficient quantityfrom plasmid-bearing cells to degrade rapidly the ampicillin in themedium (Kemp and Britz, 1987; Pierce and Gutteridge, 1985). Theresultant removal of selection pressure allows the plasmid-free cells todominate the culture (Nishimura et al., 1987). The use of APH shouldlimit the extracellular clearance of antibiotic from the medium.

The main genetic tool used to boost stability of plasmid pMJR1750 is theparB locus isolated from the multiple resistance factor R1 by K. Gerdes(1988). This locus stabilizes plasmids in a population by encoding acell-killing gene (hok) whose mRNA is activated only when the cell losesthe plasmid. Upon loss of the plasmid, the 52 amino acid Hok protein isexpressed, and the cell is rapidly killed due to a collapse of thetransmembrane potential and cessation of respiration. Therefore,although this locus does not change the rate of appearance ofplasmid-free segregants, it kills them as they are generated. The parBlocus is the only system known which utilizes this post-segregationalkilling mechanism.

The E. coli strain AMA1004 (Casadaban et al., 1983) was chosen as asuitable host. Its genotype is: Δ(lacIPOZ)C29 lacY+hsdR galU galKstrA_(T) leuB6 trpC9830 with the result that AMA1004 cannot produceβ-galactosidase due to a stable deletion, but it can produce13-galactoside permease. Hence, this strain can be used convenientlywith MacConkey agar plates to indicate cells harboring plasmidsexpressing lacZ. Plasmid-bearing cells form red colonies whereasplasmid-free cells form white colonies.

The tightly-regulated expression vector pMJR1750 (FIG. 1) capable ofproducing large quantities of β-galactosidase was obtained from Stark(1987). It includes the strong tac promoter upstream of theβ-galactosidase gene as well as the laclQ gene for complete repression.Multiple copies of the tac promoter arising from multiple copies of theplasmid can titrate the repressor if laclQ is not placed on theexpression vector (Stark, 1987). It follows that by adding thenon-cleavable lactose analog IPTG to the fermentation medium,transcription of lacZ can be induced over a wide range. This vector hasthe additional advantage that its DNA sequence has been completelydetermined.

In order to limit homologous recombination, P1 kc generalizedtransduction was used to introduce a recA deletion into the host strainAMA1004. The donor strain for the transduction was constructed by Iharaet a!. (1985) and consists of the host JC10289 with the genotypeΔ(srl-recA)306::TnlO, and the helper plasmid pKY102 which is recA+ andampicillin-resistant. The method of Silhavy et al. (1984) was used toperform the transduction of AMA1004 and consists of forming a P1 lysatefrom the donor strain and transferring this lysate to the recipientstrain.

Transductants were selected by tetracycline resistance (15 μg/mL) on LBplates and scored for ultraviolet light sensitivity. The UV sensitivitytest involved shining UV light at 260 nm (UVP Inc. Model R-526) from adistance of 3.1 cm from colonies on LB plates for periods of 5, 10, or15 seconds. The transductant colonies were checked for growth andcompared to recA− and recA+ controls that were also irradiated. Theresulting recA deletion strain derived from AMA1004 was named BK6.

In order to obtain the parB locus, pKG1022 (Gerdes, 1988) was isolatedfrom CSHSO using chloramphenicol amplification and CsCl centrifugationas described previously (Wood and Peretti, 1989). The recipient vectorpMJR1750 was isolated from AMA1004 following transformation with DNAsupplied by Stark. The parB locus was excised from pKG1022 along withaphA (Kan′) using Hindii (Boeh. Mann.), creating a DNA fragment of 1630bp. The recipient plasmid pMJR1750 (7550 bp) was digested with ScaI(Boeh. Mann.) at a unique site, abolishing ampicillin resistance. Thetwo restricted plasmids (2.5 μg each) were dialyzed and blunt-endligated (Rodriguez and Tait, 1983) using T4 DNA ligase (BRL). BK6 wastransformed with the ligated DNA following the method of Maniatis et al.(1982).

The genetic manipulations of the host/plasmid system is extremelyeffective in enhancing plasmid stability. The impact of introducing therecA mutation is illustrated by the reduction in the frequency at whichplasmid-free segregants arise (p) for the two hosts bearing pMJR1750.Under non-inducing conditions, a 33-fold decrease in p was observed forBK6 (recA−) as the host relative to AMA1004 (recA+). The relativedecrease in p due to the recA deletion was only five-fold upon IPTGinduction of lacZ. The recA mutation may enhance segregational stabilitysignificantly by limiting the formation of plasmid multimers, which formreadily in recA+ hosts (Bedbrook and Ausubel, 1976). Plasmidmultimerization has been shown to increase segregational instability bydecreasing the number of partitioning molecules (Summers and Sherratt,1984).

A dramatic increase in plasmid stability occurred upon addition of theparB locus to the expression vector. In the absence of antibioticselection pressure, p decreased by over ten orders of magnitude for bothinduced and non-induced conditions. Furthermore, chemostat stabilityincreased to 100% at all levels of transcription induction in thepresence of kanamycin using pTKW106. The original expression system,AMA1004/pMJR1750, was unstable in the chemostat even with the continuousaddition of ampicillin at 400 μg/L (1.75% plasmid-bearing at 0.5 mMIPTG). This result underscores the inadequacy of the β-lactamase gene asan effective selective marker for unstable plasmids.

True partitioning loci, which aid stabilization of plasmid maintenanceby increasing the fidelity of plasmid partitioning, have not provedfully effective in stabilizing high expression vectors (Skogman et al.,1983). In contrast, the parB system, which actively enforces plasmidstability through the post-segregational killing of any plasmid-freesegregants that arise, effectively stabilized a fully-induced expressionvector directing production of 15-20% of the total cell protein. Thisresult is highly significant since it illustrates the potential of parBas a stabilizing element in large-scale production processes whereantibiotic addition is undesirable due to its expense and the resultingcontamination of the product stream (Ensley, 1985).

The genetic stabilization strategy employed in this work can be appliedto any E. coli host/vector system. The recA mutation can be convenientlyintroduced into the host using P1 transduction, and plasmids with parBcassettes which allow easy manipulation of the parB gene. This allowedus to replace the -lactamase gene with the genes for kanamycinresistance and parB in a single step. Furthermore, the parB locus to bean effective plasmid-stabilizing element in a number of gram-negativestrains, increasing the generality of this approach.

In addition, the host/plasmid system is a very attractive model systemfor kinetic studies of the impact of cloned-gene expression on cellmetabolism. Gene expression of an easily assayed enzyme,β-galactosidase, can be varied almost 500-fold in a stable chemostatculture by varying IPTG concentrations in the medium. This allowsmolecular level responses (mRNA synthesis, ribosome population size,macromolecular stability) to be analyzed over a wide range of conditionsin an effort to determine the metabolic processes most significantlyaffected by cloned-gene expression.

The present disclosure provides compositions comprising a non-pathogenicmicroorganism comprising: a first nucleic acid sequence encoding anenzyme, or functional fragment thereof; a second nucleic acid sequenceencoding an polarization protein, or functional fragment thereof; and athird nucleic acid sequence encoding a toxin/antidote combination;wherein the enzyme, or functional fragment thereof, catalyzes excitationof a portion of a substrate or the release of a portion of a substratewhen the substrate is present in the blood of a subject; and wherein thepolarization protein facilitates the inclusion of both the first andsecond nucleic acid sequences in the non-pathogenic microorganism andits progeny upon cellular division. The present disclosure providescompositions comprising a non-pathogenic microorganism comprising: afirst nucleic acid sequence encoding an enzyme, or functional fragmentthereof; a second nucleic acid sequence encoding an polarizationprotein, or functional fragment thereof; and a third nucleic acidsequence encoding a toxin/antidote combination; wherein the enzyme, orfunctional fragment thereof, catalyzes excitation of a portion of asubstrate or the release of a portion of a substrate when the substrateis present in the blood of a subject; and wherein the polarizationprotein facilitates the inclusion of the first, second, or third nucleicacid sequences in the non-pathogenic microorganism and its progeny uponcellular division.

In some embodiments, the non-pathogenic microorganism is a probiotic. Insome embodiments, the non-pathogenic microorganism is a Gram-negativebacteria. In some embodiments, the probiotic bacteria is chosen fromEscherichia spp., Firmicutes spp., Bacteroidetes spp., Lactobacillusspp., Bifidobacteria spp., or Acidopholus spp. In some embodiments, theprobiotic is selected from Lactobacillus, Bifidobacteria, andAcidopholus. In some embodiments, the bacteria is harvested from a humanor animal sample and transformed as described herein. with our system.The samples may be from stool samples (probiotics) or human/mouse tumorsamples (bacteria that have potential to be very tumor selective). Insome embodiments, the non-pathogenic microorganism is E. coli Nissle1917 (EcN). In some embodiments, the non-pathogenic microorganism is aGram-negative bacteria but excludes one or more of the bacterial specieslisted in Table 1.

In some embodiments, genetic modification of microorganism strains,could take place by transformation with plasmids, integrate into thechromosomes, and create mutations that make them less virulent orimmunogenic. The possible mutations could be in amino acid synthesis(aroA, purI), virulence control (phoPQ), or in immunogenecity of LPS(msbB mutant). In some embodiments, the non-pathogenic microorganismcomprises a fifth nucleic acid sequence that encodes a virulence factor.In some embodiments, the non-pathogenic microorganism comprises a fifthnucleic acid sequence that encodes a mutated gene chosen from: aroA,purI, phoPQ, msbB.

In some embodiments, the first nucleic acid sequence encoding an enzyme,or functional fragment thereof, is any enzyme, or functional fragment ofthe enzyme, that is capable of catalyzing excitation of a portion of asubstrate or cleaving a portion of a substrate when the substrate ispresent in the blood of a subject. In some embodiments, the firstnucleic acid sequence encoding an enzyme, or functional fragmentthereof, is any enzyme, or functional fragment of the enzyme, that iscapable of catalyzing excitation of a portion of a substrate or cleavinga portion of a substrate when the substrate is in the presence of theenzyme. In some embodiments, the enzyme, or functional fragment thereof,is chosen from beta-galactosidase (lacZ) and the like. Such enzymesystems are well known in the art. In some embodiments, the enzyme, orfunctional fragment thereof, is capable of catalyzing excitation of aportion of a substrate or cleaving a portion of a substrate so that thesubstrate or portion of substrate thereby emits a wavelength of lightdetectable by human eye under conditions sufficient to excite thesubstrate or portion thereof. In some embodiments, the substrate emits avisible wavelength of light when exposed to white light. In someembodiments, the substrate emits a wavelength of light visible to thehuman eye when exposed to white light. In some embodiments, the secondnucleic acid sequence encodes a polarization protein, or functionalfragment thereof, wherein the polarization protein facilitates theinclusion of the first, second, and third nucleic acid sequences in thenon-pathogenic microorganism and its progeny upon cellular division. Insome embodiments, the polarization protein is a cytoplasmic microtubuleassociated protein. In some embodiments, the polarization protein isalpA (see, Example 3 for sequence). Polarization protein systems andtheir use are well known in the art. A phylogenetic search was conductedand uncovered more than 35 highly divergent families of actin-likeproteins (Alps) in bacteria. Their genes are found primarily on phagegenomes, on plasmids and on integrating conjugative elements, and arelikely to be involved in a variety of functions. Three Alps werecharacterized and all form filaments in the cell. The filaments ofAlp7A, a plasmid partitioning protein and one of the most divergent ofthe Alps, display dynamic instability and also treadmill. Alp7A requiresother elements from the plasmid to assemble into dynamic polymers in thecell. Most if not all of the Alps are indeed actin relatives, and thatactin is very well represented in bacteria.

In some embodiments, the third nucleic acid sequence encoding atoxin/antidote combination is chosen from Hok (toxin) and Sok(antidote); fst (toxin) and RNAII (antidote); TisB (toxin) and IstR(antidote); LdrD (toxin) and RdID (antidote); FlmA (toxin) and FlmB(antidote); Ibs (toxin) and Sib (antidote); TxpA/BrnT (toxin) and RatA(antidote); SymE (toxin) and SymR (antidote); XCV2162 (toxin) andptaRNA1 (antidote). These systems and their use are well known to theskilled artisan. The hok/sok system of plasmid R1, which mediatesplasmid stabilization via killing of plasmid-free segregants, encodestwo genes: hok and sok. The hok gene product is a potent cell-killingprotein. The expression of hok is regulated post-transcriptionally bythe sok gene-encoded repressor, an antisense RNA complementary to thehok mRNA leader region. The sok half-life is short lived, thus cellsmust maintain the plasmid to produce a viable amount of antidote for thehok protein.

The present disclosure also provides kits comprising the non-pathogenicmicroorganism compositions described herein. In some embodiments, thekit further comprises a substrate for the enzyme, or functional fragmentthereof. In some embodiments, the kit further comprises at least a firstcontainer comprising a rehydration solution and, optionally, a syringeand/or needle. Kits may also be supplied with instructional materials.Instructions may be printed on paper or other substrates, and/or may besupplied as an electronic-readable medium, such as a floppy disc,CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readablememory storage device. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an internetweb site specified by the manufacturer or distributor of the kit, orsupplied as electronic mail.

The present disclosure also provides kits comprising a first containercomprising any one or combination of non-pathogenic microorganismcompositions described herein; and a second container comprising anyeffective amount of substrate disclosed herein; and optionally adetection device capable of detecting the presence, absence, or quantityof UV, infrared, or visible light emitted by a sample. The presentdisclosure also provides kits comprising a first container comprisingany one or combination of non-pathogenic microorganism compositionsdescribed herein; and a second container comprising any effective amountof substrate disclosed herein; and optionally a detection device capableof detecting the presence, absence, or quantity of UV, infrared, orvisible light emitted; and optionally a third container for urine samplecollection. In some embodiments, the kits comprising a detection devicethat measures the intensity of light from a sample.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof. In some embodiments, the kit comprises: (i) at leastone nucleic acid molecule that comprises one or a combination of nucleicacid sequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof; and (ii) a container comprising an amount of a substratespecific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least one nucleic acid molecule that comprises one or acombination of nucleic acid sequences selected from: SEQ ID NO: 2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least one nucleic acid molecule that comprises one or acombination of nucleic acid sequences selected from: SEQ ID NO: 3, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least one nucleic acid molecule that comprises one or acombination of nucleic acid sequences selected from: SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof. In some embodiments, the kitcomprises: (i) at least one nucleic acid molecule that comprises one ora combination of nucleic acid sequences selected from: SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:10, and SEQ ID NO:11, and/or functional fragments thereof; and (ii) acontainer comprising an amount of a substrate specific for any of theenzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof. In some embodiments, the kitcomprises: (i) at least one nucleic acid molecule that comprises one ora combination of nucleic acid sequences selected from: SEQ ID NO: 2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, and SEQ ID NO:11, and/or functional fragments thereof; and (ii) acontainer comprising an amount of a substrate specific for any of theenzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof. In some embodiments, the kitcomprises: (i) at least one nucleic acid molecule that comprises one ora combination of nucleic acid sequences selected from: SEQ ID NO: 3, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, and SEQ ID NO:11, and/or functional fragments thereof; and (ii) acontainer comprising an amount of a substrate specific for any of theenzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstnucleic acid molecule comprising at least one or a combination ofnucleic acid sequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11, and/or functional fragments thereof. In someembodiments, the kit comprises: (i) at least one nucleic acid moleculethat comprises one or a combination of nucleic acid sequences selectedfrom: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof; and (ii) a container comprising an amountof a substrate specific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof; and wherein the second nucleic acid molecule comprises at leastone or a combination of nucleic acid sequences selected from: SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof. In some embodiments, the kitcomprises: (i) at least two nucleic acid molecules, wherein one of thenucleic acid molecules comprises one or a combination of nucleic acidsequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof; and wherein at least one additional nucleic acid moleculecomprises one or a combination of nucleic acid sequences selected from:SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, andSEQ ID NO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein the second nucleic acid moleculecomprises at least one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof. In some embodiments, the kit comprises: (i) at least twonucleic acid molecules, wherein one of the nucleic acid moleculescomprises one or a combination of nucleic acid sequences selected from:SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:10, and SEQ ID NO:11, and/or functional fragments thereof; andwherein at least one additional nucleic acid molecule comprises one or acombination of nucleic acid sequences selected from: SEQ ID NO: 2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein the second nucleic acid moleculecomprises at least one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof. In some embodiments, the kit comprises: (i) at least twonucleic acid molecules, wherein one of the nucleic acid moleculescomprises one or a combination of nucleic acid sequences selected from:SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:10, and SEQ ID NO:11, and/or functional fragments thereof; andwherein at least one additional nucleic acid molecule comprises one or acombination of nucleic acid sequences selected from: SEQ ID NO: 3, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and (ii) a containercomprising an amount of a substrate specific for any of the enzymesdisclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein the second nucleic acid moleculecomprises at least one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof. In some embodiments, the kit comprises: (i) at leasttwo nucleic acid molecules, wherein one of the nucleic acid moleculescomprises one or a combination of nucleic acid sequences selected from:SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof; and wherein at least one additional nucleic acid moleculecomprises one or a combination of nucleic acid sequences selected from:SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functional fragmentsthereof; and (ii) a container comprising an amount of a substratespecific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof; and wherein the second nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least two nucleic acid molecules, wherein one of the nucleic acidmolecules comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein at least one additional nucleic acidmolecule comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and (ii) a container comprising an amount of asubstrate specific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof; and wherein the second nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least two nucleic acid molecules, wherein one of the nucleic acidmolecules comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein at least one additional nucleic acidmolecule comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and (ii) a container comprising an amount of asubstrate specific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof; and wherein the second nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/orfunctional fragments thereof. In some embodiments, the kit comprises:(i) at least two nucleic acid molecules, wherein one of the nucleic acidmolecules comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and wherein at least one additional nucleic acidmolecule comprises one or a combination of nucleic acid sequencesselected from: SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and/or functionalfragments thereof; and (ii) a container comprising an amount of asubstrate specific for any of the enzymes disclosed herein.

The present disclosure also provides a kit comprising at least a firstand a second nucleic acid molecules; wherein the first nucleic acidmolecule comprises at least one or a combination of nucleic acidsequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and wherein the secondnucleic acid molecule comprises at least one or a combination of nucleicacid sequences selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof. In some embodiments, the kitcomprises: (i) at least two nucleic acid molecules, wherein one of thenucleic acid molecules comprises one or a combination of nucleic acidsequences selected from: SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, and/or functional fragments thereof; and wherein at least oneadditional nucleic acid molecule comprises one or a combination ofnucleic acid sequences selected from: SEQ ID NO:2, SEQ ID NO: 3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11, and/or functional fragments thereof; and (ii) acontainer comprising an amount of a substrate specific for any of theenzymes disclosed herein.

The present disclosure also provides methods of diagnosing a tumor in asubject comprising: i) administering a non-pathogenic microorganismcomposition described herein to a subject in an effective amount; ii)administering a substrate for the enzyme, or functional fragmentthereof, to the subject in an effective amount; and iii) detecting thepresence or absence of the released or excited portion of the substratein the urine of the subject.

The present disclosure also provides methods of diagnosing a metastatictumor in a subject comprising: i) administering a non-pathogenicmicroorganism composition described herein to a subject in an effectiveamount; ii) administering a substrate for the enzyme, or functionalfragment thereof, to the subject in an effective amount; and iii)detecting the presence or absence of the released or excited portion ofthe substrate in the urine of the subject.

In some embodiments, the non-pathogenic microorganism compositiondescribed herein is administered to the subject in the form of a dosageform such as a pill, tablet, or a capsule, or as a food product such asyogurt. In some embodiments, the composition of any one of claims 1 to12 is administered to the subject per os. In some embodiments, thesubstrate is administered per intravenous injection.

In some embodiments, the substrate for the enzyme, or functionalfragment thereof, is administered to the subject via a dosage form orvia intravenous administration. The identity of the substrate dependsupon the enzyme system selected. In some embodiments, the substrate ischosen from S-Gal, Ch-Red, and LuGal.

The cleaved portion of the substrate or excitation of a portion of thesubstrate as acted upon by the enzyme is detected in the urine of thesubject. In those subjects having a tumor, administration of thenon-pathogenic microorganism composition causes a colonization of thetumor by the non-pathogenic microorganism, whereby, rather than dying ifno tumor is present, these non-pathogenic microorganism actuallyproliferate and expand in numbers, resulting in their presence after 48hours upon administration. Thus, measurement of the presence or absenceof the enzymatic activity (i.e., cleavage product or excitation) after a48 hour period is indicative of the presence of a tumor. In someembodiments, the presence or absence of the released or excited portionof the substrate is determined by identifying a change in the color ofthe urine. In some embodiments, a period of time is allowed to elapseafter step i) sufficient for colonization of the microorganism in atumor cell, tumor tissue, or a cell associated with a hyperproliferativedisorder. In some embodiments, the quantity of the released or excitedportion of the substrate is determined by measuring fluorescence oremission of visible wavelengths of light when exposed to white light. Insome embodiments, the quantity of the released or excited portion of thesubstrate is determined by colorimetric analysis.

In some embodiments, when the substrate is SGal, the presence or absenceof the released or excited portion of the substrate is determined bycontacting a urine sample from the subject to iron ions. In someembodiments, when the substrate is LuGal, the presence or absence of thereleased or excited portion of the substrate is determined byquantifying the amount of luciferin in the urine sample of the subject.

In some embodiments, the tumor is derived from the gastrointestinaltract or urinary system of the subject. In some embodiments, the cancercell, cancer tissue or cell associated with a hyperproliferativedisorder is a cancer derived from the gastrointestinal tract or urinarysystem of a subject.

In some embodiments, the presence or absence of the released or excitedportion of the substrate is determined by quantifying the amount ofreleased or excited portion of the substrate present in the subject atone or a plurality of sites in the subject. In some embodiments,

The present disclosure also provides methods of quantifying the numberof cancer cells in a cell sample comprising: i) contacting the cellsample with a non-pathogenic microorganism composition described hereinto form a mixture; ii) exposing the mixture to a substrate for theenzyme, or functional fragment thereof; and iii) measuring the amount ofthe released or excited portion of the substrate in the mixture. Thegreater the amount of released or excited portion of the substrate, thegreater the number of non-pathogenic microorganism and, hence, thegreater number of tumor cells.

The present disclosure also provides methods of detecting a cancer cell,cancer tissue, or cell associated with a hyperproliferative disorder ina subject comprising: i) administering non-pathogenic microorganismcomposition described herein to the subject; ii) administering asubstrate for the enzyme, or functional fragment thereof, to thesubject; and iii) detecting the presence or absence of the releasedportion of the substrate.

The present disclosure also provides any one or more of the foregoingnon-pathogenic microorganism compositions described herein for detectingthe presence of a tumor in a subject.

The present disclosure also provides any one or more of the foregoingnon-pathogenic microorganism compositions described herein for use inthe manufacture of a product for detecting the presence of a tumor in asubject.

The present disclosure also provides uses of any one or more of theforegoing non-pathogenic microorganism compositions described herein fordetecting the presence of a tumor in a subject.

The present invention also provides uses of any one or more of theforegoing non-pathogenic microorganism compositions described herein inthe manufacture of a medicament for detecting the presence of a tumor ina subject.

In some embodiments, the non-pathogenic microorganism is capable ofcolonization after administration within about 1, 2, or 3 days. In someembodiments, the non-pathogenic microorganism is capable of beingcleared by the immune system or body after administration within about2, 3, 4, 5, 6, or 7 days.

Methods

In some embodiments, methods are provided which use non-pathogenicmicroorganisms that comprise plasmids to colonize tumors and deliverproteins encodes by nucleic acid sequences in the bacteria. Thenon-pathogenic microorganisms that comprise plasmids comprise nucleicacid sequences that facilitate the inclusion of copies of plasmids inthe progeny upon cellular division. A much larger proportion of totalprogeny of the non-pathogenic microorganisms remain plasmid bearing isthe non-pathogenic microorganisms that comprise plasmids comprisenucleic acid sequences that facilitate the inclusion of copies ofplasmids in the progeny upon cellular division compared to theproportion of plasmid bearing non-pathogenic microorganisms among thetotal progeny of the non-pathogenic microorganisms that compriseplasmids but that do not comprise the nucleic acid sequences thatfacilitate plasmid copy inclusion. In some embodiments, the nucleic acidsequences that facilitate plasmid copy inclusion include nucleic acidsequences that encode polarization protein. Polarization proteins arepreferably cytoplasmic microtubule associated protein. In someembodiments, the nucleic acid sequences that facilitate plasmid copyinclusion include nucleic acid sequences that encode actin-like proteins(Alps). In some embodiments, the Alp may be AlpA or Alp7a. In someembodiments, the nucleic acid sequences that facilitate plasmid copyinclusion include nucleic acid sequences that encode a bacterial toxinand its antidote. In some embodiments, any of the many toxin-antidotecombinations known in the art and/or disclosed herein may be provided.In some embodiments, the toxin-antidote combination hok/sok is provided.In some embodiment, both nucleic acid sequences that encode polarizationprotein and nucleic acid sequences that encode a bacterial toxin and itsantidote are provided in the non-pathogenic microorganisms that compriseplasmids as the inclusion of these two elements within a non-pathogenicmicroorganism that comprises plasmids provides a higher level comparedto level of plasmid-bearing progeny among total progeny ofnon-pathogenic microorganisms that comprise plasmids and that compriseone but not both of these two elements, much less the level ofplasmid-bearing progeny among total progeny of non-pathogenicmicroorganisms that comprise plasmids and that comprise neither element.A single element provides greater plasmid retention/lower plasmid lossamong progeny compared to no element. Two elements provides greaterplasmid retention/lower plasmid loss among progeny compared to a singleelement.

In methods comprising the use of non-pathogenic microorganisms thatcomprise plasmids and that comprise nucleic acid sequences encoding apolarization protein and/or nucleic acid sequences encoding atoxin-antidote combination to colonize tumors in a subject, proteinproducts encoded by nucleic acid sequences on plasmids are provided bethe non-pathogenic microorganisms in the tumor for a longer duration doto the longer period of plasmid retention among generations of progenymaking such methods more effective in producing proteins encoded bynon-pathogenic microorganisms in tumors. In methods in which thenon-pathogenic microorganisms that comprise plasmids are used to deliverproteins involved in therapeutic processes or effects, non-pathogenicmicroorganisms that comprise plasmids which encode therapeuticallyrelevant proteins and that comprise nucleic acid sequences encoding apolarization protein and/or nucleic acid sequences encoding atoxin-antidote combination to colonize tumors in a subject provideimprovements over similar methods in which the non-pathogenicmicroorganisms comprise neither nucleic acid sequences encoding apolarization protein nor nucleic acid sequences encoding atoxin-antidote combination. Therapeutically relevant proteins expressedfrom plasmids in non-pathogenic microorganisms that colonize tumors in asubject are known and the methods are improved as disclosed hereinthrough the provision of nucleic acid sequences encoding a polarizationprotein and/or nucleic acid sequences encoding a toxin-antidotecombination to effect longer plasmid retention among generations ofprogeny.

Likewise, in methods in which the non-pathogenic microorganisms thatcomprise plasmids are used to deliver proteins involved in diagnosticprocesses or effects, non-pathogenic microorganisms that compriseplasmids which encode diagnostically relevant proteins and that comprisenucleic acid sequences encoding a polarization protein and/or nucleicacid sequences encoding a toxin-antidote combination to colonize tumorsin a subject provide improvements over similar methods in which thenon-pathogenic microorganisms comprise neither nucleic acid sequencesencoding a polarization protein nor nucleic acid sequences encoding atoxin-antidote combination. Diagnostically relevant proteins expressedfrom plasmids in non-pathogenic microorganisms that colonize tumors in asubject are known and the methods are improved as disclosed hereinthrough the provision of nucleic acid sequences encoding a polarizationprotein and/or nucleic acid sequences encoding a toxin-antidotecombination to effect longer plasmid retention among generations ofprogeny. In some embodiments, the diagnostically relevant proteins arethose encoded by nucleic acid sequences on plasmids are described hereinsuch as enzymes which process substrates that can be used in analysis ofurine to determine if enzymes are present in the subject, indicating thepresence of tumors colonized by the non-pathogenic microorganism. Suchmethods are described herein and involve either the detection in asubject's urine of products of enzyme processing of a substrateindicating the presence of enzyme, or the detection in reduced levels ina subject's urine of unprocessed substrate indicating an increase insubstrate processing and thus the presence of enzyme. Typically, in suchmethods, the substrate is administered to the subject after time haselapsed for colonization by the non-pathogenic microorganism in a tumorpresent in a subject. Some methods provide a color change of urine whenit contains processed substrate products, the color being directlyprovided by the processed substrate product. Some methods provide acolor change of urine when it contains substrate products, the colorbeing provided reactions that occur if processed substrate products arepresent. For example, methods may employ antibodies which bind toprocessed substrate products but not unprocessed substrate. Suchantibodies may be contacted with a sample of urine and if processedsubstrate products are present, an antibody-processed substrate productcomplex forms. Detection of the antibody-processed substrate productcomplex indicated the presence of processed substrate product in thesample which indicates the presence of the enzyme produced bynon-pathogenic microorganisms colonizing a tumor.

In some embodiments, the administration of non-pathogenic microorganismcomprises a signal to noise ratio of no less than about 2, about 3,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9, about 4.0, about 4.1, or about 4.2.

TABLE 1 Non-pathogenic Microorganisms Species Plasmid GenBank Accessionno. B. longum pNAC2 AY112723.1 pTB6 NC_006843.1 pB44 AY066026.1 pKJ36AF139129.1 pMG1 NC_006997.1 pBLO1 AF540971.1 p6043B DQ458911 pNAC1AY112724.1 pNAL8L AM183145.1 pKJ50 U76614.1 pNAL8M AM183144.1 pBIFA24NC_010164.1 p6043A DQ458910 pNAC3 AY112722.1 pDOJH10L AF538868.1pDOJH10S AF538869.1 pMB1 X84655 pSP02 GU256055.1 pFI2576 NC_011139.1BLNIAS_P1 CP002795.1 BLNIAS_P2 CP002796.1 p157F-NC1 AP010891.1 P157F-NC2AP010892.1 pBK283 AB495342.1 B. breve pCIBb1 AF085719.1 pNBb1 E17316pB21a NC_010930.1 B. pseudolongum subsp. pASV479 NC_010877.1 glohosum B.bifidum pB80 NC_011332.1 pBIF10 DQ093580 B. asteroides pCIBAO89NC_010908.1 pAP1 Y11549 B. catenulatum pBC1 NC_007068.1 B.pseudocatenulatum p4M AF359574.1 Lactococcus ssp Lactobacilli sspLactobacilli fermentum, Lactobacilli acidophilus, Lactobacilli caseiLactobacilli plantaru Attenuated pathogens (known delivery vectors forDNA/antigens): E. coli Shigella spp Salmonella spp Salmonella (allsubstrains, including attenuated strains for tumor experiments, VNP20009human clinical strain) Listeria spp Mycobacteria spp Pseudomonas sppBordetella spp Bacillus spp including B. subtilis StaphylococciStreptococci Enterobacter spp Enterococci spp Acinetobacter spp Vibriospp. Pasteurella spp. Burkholderia spp Other Pseudomonas sppEdwardsiella spp Erwinia spp Shewanella spp (mosquito pathogen)Klebsiella spp Chlamydia spp Aeromonas spp Plant pathogens, optionallyattenuated Agrobacterium spp. Rhizobium spp Ralstonia spp Xanthomonasspp. Geobacter spp Photobacteria spp

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. In order that theinvention disclosed herein may be more efficiently understood, examplesare provided below. It should be understood that these examples are forillustrative purposes only and are not to be construed as limiting theinvention in any manner Throughout these examples, molecular cloningreactions, and other standard recombinant DNA techniques, were carriedout according to methods described in Maniatis et al., MolecularCloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989),using commercially available reagents, except where otherwise noted. Alljournal articles, patent applications, issued patents or other citationsdisclosed in this document are incorporated by reference herein in theirentireties.

TABLE 2 Hok sequence schematic C BamHIAACAAACTCCGGGAGGCAGCGTGATGCGGCAACAATCACACGGATTTCCCGTGAACGGTCTGAATGAGCGGATTATTTTCAGGGGAAAGTGAGTGTGGTCATTGTTTGAGGCCCTCCGTCGCACTACGCCGTTGTTAGTGTGCCTAAAGGGCACTTGCCAGACTTACTCGCCTAATAAAAGTCCCTTTCACTCACACCAGTphok hok mRNA101                                                                                   200GCGTGCAGGTATATGGGCTATGATGTGCCCGGCGCTTGAGGCTTTCTGCCTCATGACGTGAAGGTGGTTTGTTGCCGTGTTGTGTGGCAGAAAGAAGATACGCACGTCCATATACCCGATACTACACGGGCCGCGAACTCCGAAAGACGGAGTACTGCACTTCCACCAAACAACGGCACAACACACCGTCTTTCTTCTAT                                               mok start201                                                                                            300GCCCCGTAGTAAGTTAATTTTCATTAACCACCACGAGGCATCCCTAIGTCTAGTCCACATCAGGAYAGCCTCTTACCGCGCTTTGCGCAAGGAGAAGAAGCGGGGCATCATTCAATTAAAAGTAATTGGTGGTGCTCCGTAGGGATACAGATCAGGTGTAGTCCtATCGGAGAATGGCGCGAAA CTCTTCTTCJ -10 psok -35 sok RNA hok start301                                        SaU3A                                               400GCCAAACTACCACGAAGTTCCCTTGTCTGGTGTGTGTTGATCGTGTGTCTCACACTGTTGATATTCACTTATCTGACACGAAAATCGCTGTGCGAGACGGTACTTTGATGGTGCTTCAAGGGAACAGACCACACACAACTAGCACACAGAGTGTGACAACTATAAGTGAATAGACTGTGCTTTTAGCGACACGCTCTIV401                                                                                           500TTCGTTACAGAGAtGGACACAGGGAGGTGGCGGCTTTCATGGCTTACGAATCCGGTAAGIAQCACCTGGAGGCGGGCGCAGGCCCGCCTTTTCAGGACTAAGCAATGTCTCTGCCTGTGTCCCTCCACCGCCGAAAGTACCGAATGCTTAGGCCATTCATCGTTGGACCTCCGCCCGCGTCCGGGCGGAAAAGTCCTGAmok hok                                                                    Termination of                                                                    truncated mRNA580GATGCTGGTCTGACTACTGAAGCGCCTTTATAAAGGGGCTGCTGGTTCGCCGGTAGCCCCTTTCTCCTTGCTGATGTTGTEcoRICTACGACCAGACTGATGACTTCGCGGAAATATTTCCCCGACGACCAAGCCc rATCGGGGAAAGAGGAACGACTACAACA                                                            Termination of full length                                                            hok transcript A

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EXAMPLES Example 1: Preparing Electrocompetent Cells andElectroporations

To make about 7-8 aliquots of electro-competent cells we performed thefollowing steps: 1) grow 5 mL strain of interest with pKD46 at 30 Covernight; 2) prepare two flasks with 1/100× dilution of overnight in250 mL LB and grow at 30 C; 3) label 1 flask+and the other—L-arabinose(control); 4) when the OD₆₀₀ of the cells (+pKD46) reaches 0.1 (about1-1.5 hours for JS006-24 minutes doubling), we added L-arabinose toconcentration of 0.15-1.5% to induce pKD46 λ-red expression; i) we addedabout 2 mL of 25% L-arabinose to 250 mL+culture, none to − culture; 5)we continued to grow the culture at 30° C. to OD₆₀₀=0.4 (about 1-2hours); 6) chill cells in ice-water bath 10 minutes; 7) centrifuge 10minutes at 4000 rcf 4° C. in 35 mL nalgene centrifuge tubes (or usesterile 50 mL tubes, max spin 4 g); 8) pipette off supernatant andresuspend pellets in 1-5 mL ice-cold dH₂O (filtered); 9) centrifuge minat 4000 rcf 4° C.; 10) pipette off all dH₂O carefully; 11) optionally,perform another spin wash step in ice-cold dH₂0; 12) resuspend pellet in1000 μL dH₂0+10-15% glycerol; and 13) aliquot 50 uL per tube(prechilled).

Example 2: Electroporation for Insertion

For electroporation step, two conditions are included: +/−PCR fragment.Electroporation cuvettes are chilled for 5 minutes on ice (or don't needif kept in −20 C). 5 μg to 0.5 μg of PCR amplified DNA are added tocells (for genomic insertions: typically add 50-100 ng (of 50-100ng/μL)). Electroporation apparatus is set to “Bacteria”. 1 mL SOC isprepared in pipette. Take cuvette off ice, wipe metal electrodes withkimwipe. Place the cuvette into the sample chamber (quickly so nocondensation on electrodes occurs). Apply the pulse by pushing thebutton. Remove the cuvette. Immediately add 1 mL LB medium and transferto a sterile culture tube. Incubate 60-120 min with moderate shaking at37° C. Plate at 37° C. (for genomic insertions). If transformationdoesn't work, replate in the morning (as in Datsenko procedure) afterlonger incubation period.

For in vitro analysis, a proxy of lacZ activity expression from plasmidswas used as a marker for presence of the plasmid. Cells were grownovernight without antibiotics, where presumably many cells without astability lose their plasmid and ability to produce lacZ. As observed,both no stability or hok systems lose expression of lacZ under inducingconditions, while the hok+alpA combined system retains significantactivity.

In vivo data highlights the stability of this system. Typically 90% ofbacteria lose their plasmids within 24 hours, and slightly less for the“stabilized” hok system. With hok+alpA, about 100% maintenance over thecourse of 2-3 days and significant maintenance over the course of a weekwas observed.

Example 3: Compositions Comprising Nucleic Acids Encoding Enzyme withoutAdministration of Substrate to Subject (PROPHETIC)

A secreted enzyme can be used to achieve a better signal to noise ratio,due to cleavage of the injected substrate in the body, however, enzymesare generally too large to clear the renal filtration limit and reachthe bladder. Any of the above methods to construct the aforementionedplasmids and bacterial colonies may be conducted except that the methodsdo not necessarily have to include the step of administering a substrateto a subject if an enzyme product produced by the non-pathogenicmicroorganism is of a size sufficient to pass through the renalfiltration system of the kidneys and enter the urine of a subject towhom a composition comprising the non-pathogenic microorganism has beendelivered. In such a system, the component enzyme will comprise anucleic acid sequence encoding one component from a two partalpha-complementation scheme of a marker, such as beta-galactosidase.With this new scheme, a small piece of enzyme, known as the alphafragment will be expressed in bacteria from the stabilized plasmids orthe genome. Once collected in the urine, it will be mixed with purifiedomega fragments (the larger component of beta-galactosidase), toassemble a functional enzyme. Finally, substrate will be added to asample of the urine taken from a subject to whom the composition hadbeen administered to detect for the presence of the enzyme and hencepresence of the bacteria and tumor. Such a substrate may be present on astick, paper, or in a solution easily exposed to the sample.

Example 4: AlpA Sequence Incorporated into the pTKW-alpA PlasmidSequence (SEQ ID NO:1)

gaatactgtttcctgtgtgaaattgttatccgctcacaattccacacattatacgagccgatgattaattgtcatatccagaacgggagtgcgccttgagcgacacgaattatgcagtgatttacgacctgcacagccataccacagcttccgatggctgcctgacgccagaagcattggtgcaccgtgcagtcgatgataagagtcaaacatgagaattaattctgcattaatgaatcggccaacgcgcggggagaggcggtagcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcattctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaatcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcataacaaactccgggaggcagcgtgatgcggcaacaatcacacggatttcccgtgaacggtctgaatgagcggattattttcagggaaagtgagtgtggtcagcgtgcaggtatatgggctatgatgtgcccggcgcttgaggctttctgcctcatgacgtgaaggtggtttgttgccgtgttgtgtggcagaaagaagatagccccgtagtaagttaattttcattaaccaccacgaggcatccctatgtctagtccacatcaggatagcctataccgcgctttgcgcaaggagaagaaggccatgaaactaccacgaagttcccttgtctggtgtgtgttgatcgtgtgtctcacactgttgatattcacttatctgacacgaaaatcgctgtgcgagattcgttacagagacggacacagggaggtggcggctttcatggcttacgaatccggtaagtagcaacctggaggcgggcgcaggcccgccttttcaggactgatgctggtctgactactgaagcgcctttataaaggggctgctggttcgccggtagcccctttctccttgctgatgttgtgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtgaggttcttgcaggtcggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcctgaattgactacttccgggcgctatcatgccataccgcgaaaggttttgcaccacctaggtcattagcctccaatcttatagtgaaactccgcaaacttcgtttcctcaatattgggaatactcgataacttttgttcagctttttcataaaaagaaattgctttaacataattttttttaaaaaactcataatcccctgcaaacaattcaaaataaaaatttattaaaagatcatgacttcccttacttaacatattaacgacatcacccatagatgcctcttgatcaaaacaaaggctatgacgatgatccagtagaataaaataatcaaccaggtccttattatcttcagcctgacttaacaattgctttatttcagttcttttctctatagactcatgaaccttctcctgcataatatagtcatgccatttattcaacagattgcctacatagggagacggtactttttttactttggacaacatacacttccctccagctattcaaaaatcaaaactagtcctaatacttatcggcatcatcaaacattttaaaacacagaacatttagtacatagtgcttaatgttcaaatctcattttgatgactgaacccaagcatgtactgatatcatactaaacggctcaaacacaatctatacaagtttttaagataggccaaagggaataacagtatacgttagtgaaatcccacaggaaaaatatattaaaatactaatgttctatcaaacgaacaacccttaaaaaggaacttaaaacctctgggttttaaggaaattcgcattttatttagtgtttttctcttgactttgagaacttgaaactagcagaatagctgactgttctaggaaacagggcgaatttcgattgcctatgtctgtcgcgcaaaaaataaaaacggacagacataggcaatcgatcaggatttgaaactagcgtcatagagacgtctgaggtttccagctctgccttgctatcgccaggctttcgcctgccatgacctttttacatacaatgcttgtcctgtatgcaacttctatggggtttgtctcgtgttctctcacacggtcacactcaattgtgtgccgctgcatagaagcttggccatagttgcccgcaccgtagtgcgccaagcaacctagtggtttatccacattctccggaccgttaatggccgtcctcgccattcaccacaagcgcagcaaggaacgcttattgtggtatatccccgggtttgcggtggacggggcaactcctgacgtcagtttattttacaccccttaacggcagctgggtgacaaacaaaaaacgacagaaaaccacggtttgataccctccaaacagtggttttctgtcgtccaaaaatagccgaaaagtgttgacgtatacacttgttttcggtaaaatgaagacataacttaaacattgtaagtgagggcttacaaaccaagtgttcgatgctgcaacatcggacactttttatttgtcattctttatttgtattcaattttgcaaatagctcgcaaacaaaatatgtatcatcaaatctattaaccttgttgtctgcaaacaacagggttttttgttgtttatttagaataactagaaccagaattcaatgccaaaactttcacattgacttaacttgactttatcttacacgattttttttttgacgtaaagccccgggcctgaaatcacttttctctactgatttcactgatttcatttttattatataatcctcaaatagcctgtattcactgattttaaatgtgatttcattttattgactttagtgatataagatgctagtattgaggaaagtgaaatcaaaggagagaataaaaatatgaatatttctcgtatgaacgtggactttggaaacagtatgtacatgaatttaattgatggttatttttttgaattgcctacaaatgtagtagagatatctaaagaagctgctgaaggaaaatttacgagtatcgttgaagatccggcagatttaaaggaccggttattagtttctacagttattgatgaaacagagagatattttctagttggtgaacttgctgaaccagaagtgttaggcaaccaacacatcaagaagttacataataaagtagagtcacatattccatacgtaacatttttagctgcaactgcttattaccaagcgctaaaaggcaaacgtgaagataatgaagttactattgaatactttcaaacaatgctaccaatttggcttcttaaaaaattggataagttcagtgaaatgcagaaaaggatggcatctaaatttttgggcactcaccaagtaaaggtgctgacattaggattagaaaaagagcttactataaaagtggaagatgcagcgtgcaggatcgaatctgaagtagcaagatgggcaataaagaaaaactttgacctagaagataaagactatgccgaacaatttaaaaattatgacgtagttttttgtgatttaggtggcggaacagatgatctagtattactaccagctggattaaaaccgccaaaaagtcgtgattatttgtttctaataccgaagcaccgtttttagcgcacttagaaaaattgagaaaagaaaaactcctagagcactttgatagcgttagggagcttgaaaagtttatatactcaaatattggaaaaactaagatggaacgaagagacgggaataccggtcagaaatttgatttaactgatatcatcaaaaaatctcttaaagaatacacagaaatcaaaatagcccaagctgaaaatacgttccctgcaccaaaagataaggtttacaaatacctttattttggcggtgttggcgaggtgcttgaagaatcaattagtgtggttactgaagagagatatggccgtgatatttctgaatcaaatcatatagttgctgaggatgcaagactgctcaacttatatggccttgaagttttaagccgcgctgaacaagtaaagaaacaggcaaatgaaaaagaggcacaatcaatttaggtgattagaaatggggaaaaacaaaagaattccactctttaatgtccgaacaacacaaatgtctgatgaaatgtacgattttgttttagagcagattagtacattcagtaaaggtaagagtaagggtacctttagagagtatgcctttcagctcatagaaagggacatgcaacaacagaaagaggaacagcaaaatagagaaaaagatcgtcatgttcatgatgaattaattgccatgagagaagaaatgaaaaaagaatttcgtgatttgaggaaaaaaattgatcagggatcgatctacgtagaacacaaaacagctgatccaaagtcagcttcaaaaacgattgaagaaggtcagttaatcactgaaaaaatcactggaactattgaagaagaatacgactatgatttttaagagcctggattaatctaggctctttttttatgccatttaagggaggattgcatgacaaactttttttagttgcaacacagacgccctgagcaaccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtattcgactgagcctttcgttttatttgatgcctctagcacttagactcgagcggccgctttttgacaccagaccaactggtaatggtagcgaccggcgctcagctggaattccgccgatactgacgggctccaggagtcgtcgccaccaatccccatatggaaaccgtcgatattcagccatgtgccttcttccgcgtgcagcagatggcgatggctggtttccatcagttgctgttgactgtagcggctgatgttgaactggaagtcgccgcgccactggtgtgggccataattcaattcgcgcgtcccgcagcgcagaccgttttcgctcgggaagacgtacggggtatacatgtctgacaatggcagatcccagcggtcaaaacaggcggcagtaaggcggtcgggatagttttcttgcggccctaatccgagccagtttacccgctctgctacctgcgccagctggcagttcaggccaatccgcgccggatgcggtgtatcgctcgccacttcaacatcaacggtaatcgccatttgaccactaccatcaatccggtaggttttccggctgataaataaggttttcccctgatgctgccacgcgtgagcggtcgtaatcagcaccgcatcagcaagtgtatctgccgtgcactgcaacaacgctgcttcggcctggtaatggcccgccgccttccagcgttcgacccaggcgttagggtcaatgcgggtcgcttcacttacgccaatgtcgttatccagcggtgcacgggtgaactgatcgcgcagcggcgtcagcagttggtttttatcgccaatccacatctgtgaaagagagcctgactggcggttaaattgccaacgcttattacccagctcgatgcaaaaatccatttcgctggtggtcagatgcgggatggcgtgggacgcggcggggagcgtcacactgaggattccgccagacgccactgctgccaggcgctgatgtgcccggcttctgaccatgcggtcgcgttcggttgcactacgcgtactgtgagccagagttgcccggcgctctccggctgcggtagttcaggcagttcaatcaactgtttaccttgtggagcgacatccagaggcacttcaccgcttgccagcggcttaccatccagcgccaccatccagtgcaggagctcgttatcgctatgacggaacaggtattcgctggtcacttcgatggtttgcccggataaacggaactggaaaaactgctgctggtgttttgcttccgtcagcgctggatgcggcgtgcggtcggcaaagaccagaccgttcatacagaactggcgatcgttcggcgtatcgccaaaatcaccgccgtaagccgaccacgggttgccgttttcatcatatttaatcagcgactgatccacccagtcccagacgaagccgccctgtaaacggggatactgacgaaacgcctgccagtatttagcgaaaccgccaagactgttacccatcgcgtgggcgtattcgcaaaggatcagcgggcgcgtctctccaggtagcgaaagccattttttgatggaccatttcggcacagccgggaagggctggtcttcatccacgcgcgcgtacatcgggcaaataatatcggtggccgtggtgtcggctccgccgccttcatactgcaccgggcgggaaggatcgacagatttgatccagcgatacagcgcgtcgtgattagcgccgtggcctgattcattccccagcgaccagatgatcacactcgggtgattacgatcgcgctgcaccattcgcgttacgcgttcgctcatcgccggtagccagcgcggatcatcggtcagacgattcattggcaccatgccgtgggtttcaatattggcttcatccaccacatacaggccgtagcggtcgcacagcgtgtaccacagcggatggttcggataatgcgaacagcgcacggcgttaaagttgactgcttcatcagcaggatatcctgcaccatcgtctgctcatccatgacctgaccatgcagaggatgatgctcgtgacggttaacgcctcgaatcagcaacggcttgccgttcagcagcagcagaccattttcaatccgcacctcgcggaaaccgacatcgcaggcttctgcttcaatcagcgtgccgtcggcggtgtgcagttcaaccaccgcacgatagagattcgggatttcggcgctccacagtttcgggttttcgacgttcagacgtagtgtgacgcgatcggcataaccaccacgctcatcgataatttcaccgccgaaaggcgcggtgccgctggcgacctgcgtttcaccctgccataaagaaactgttacccgtaggtagtcacgcaactcgccgcacatctgaacttcagcctccagtacagcgcggctgaaatcatcattaaagcgagtggcaacatggaaatcgctgatttgtgtagtcggtttatgcagcaacgagacgtcacggaaaatgccgctcatccgccacatatcctgatcttccagataactgccgtcactccagcgcagcaccatcaccgcgaggcggttttctccggcgcgtaaaaatgcgctcaggtcaaattcagacggcaaacgactgtcctggccgtaaccgacccagcgcccgttgcaccacagatgaaacgccgagttaacgccatcaaaaataattcgcgtctggccttcctgtagccagctttcatcaacattaaatgtgagcgagtaacaacccgtcggattctccgtgggaacaaacggcggattgaccgtaatgggataggtcacgttggtgtagatgggcgcatcgtaaccgtgcatctgccagtttgaggggacgacgacagtatcggcctcaggaagatcgcactccagccagctttccggcaccgcttctggtgccggaaaccaggcaaagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacgggatctatcatA full restriction map of the genetic construct with LacZ, AlpA, andsok/hok genes is depicted in FIG. 1 and is described below in Example 4.

Example 5: Plasmid pTKW106alpA

The plasmid was constructed by adding the 3.5 kb alp7AR cassette fromthe B. subtilis natto plasmid pLS20 to the pTKW106 lacZ expressionvector containing the hok/sok plasmid maintenance system. The alp7AR wasamplified by PCR and AvrII/NheI restriction sites were added usingprimers P1-2. For the purpose of cloning the alp7AR cassette intopTKW106, the entire 9 kb pTKW106 backbone was amplified and a singleAvrII restriction site was added using primers P3-4. This PCR productwas then digested with AvrII and ligated with the AvrII/NheI digestedalp7AR insert, producing pTKW106alpA.

P1: (SEQ ID NO: 12) ccacca cctagg tcattagcctccaatcttatagtg P2:(SEQ ID NO: 13) ccacca gctagc gttgctcagggcgtct P3: (SEQ ID NO: 14)ccacca cctagg cggcggatttgtcc P4: ccacComponents of the plasmid include:

colE1 sequence (SEQ ID NO: 2)gcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaa KanR sequence(SEQ ID NO: 3) gaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgt tcaatcat Hok(SEQ ID NO: 4) aacaaactccgggaggcagcgtgatgcggcaacaatcacacggatttcccgtgaacggtctgaatgagcggattattttcagggaaagtgagtgtggtcagcgtgcaggtatatgggctatgatgtgcccggcgcttgaggctttctgcctcatgacgtgaaggtggtttgttgccgtgttgtgtggcagaaagaagatagccccgtagtaagttaattttcattaaccaccacgaggcatccctatgtctagtccacatcaggatagcctcttaccgcgctttgcgcaaggagaagaaggccatgaaactaccacgaagttcccttgtctggtgtgtgttgatcgtgtgtctcacactgttgatattcacttatctgacacgaaaatcgctgtgcgagattcgttacagagacggacacagggaggtggcggctttcatggcttacgaatccggtaagtagcaacctggaggcgggcgcaggcccgccttttcaggactgatgctggtctgactactgaagcgcctttataaaggggctgctggttcgccggtagcccctttctccttgctgatgttgt alp7 (SEQ ID NO: 5)tcattagcctccaatcttatagtgaaactccgcaaacttcgtacctcaatattgggaatactcgataacttagttcagattttcataaaaagaaattgctttaacataattttttttaaaaaactcataatcccctgcaaacaattcaaaataaaaatttattaaaagatcatgacttcccttacttaacatattaacgacatcacccatagatgcctcttgatcaaaacaaaggctatgacgatgatccagtagaataaaataatcaaccaggtccttattatcttcagcctgacttaacaattgctttatttcagttcttttctctatagactcatgaaccttctcctgcataatatagtcatgccatttattcaacagattgcctacatagggagacggtactttttttactttggacaacatacacttccctccagctattcaaaaatcaaaactagtcctaatacttatcggcatcatcaaacattttaaaacacagaacatttagtacatagtgcttaatgttcaaatctcattttgatgctctgaacccaagcatgtactgatatcatactaaacggctcaaacacaatctatacaagtttttaagataggccaaagggaataacagtatacgttagtgaaatcccacaggaaaaatatattaaaatactaatgttctatcaaacgaacaacccttaaaaaggaacttaaaacctctgggttttaaggaaattcgcattttatttagtgtattctcttgactttgagaacttgaaactagcagaatagctgactgttctaggaaacagggcgaatttcgattgcctatgtctgtcgcgcaaaaaataaaaacggacagacataggcaatcgatcaggatttgaaactagcgtcatagagacgtctgaggtttccagctctgccttgctatcgccaggattcgcctgccatgacctttttacatacaatgcttgtcctgtatgcaacttctatggggtttgtctcgtgttctctcacacggtcacactcaattgtgtgccgctgcatagaagcttggccatagttgcccgcaccgtagtgcgccaagcaacctagtggtttatccacattctccggaccgttaatggccgtcctcgccattcaccacaagcgcagcaaggaacgcttattgtggtatatccccgggtttgcggtggacggggcaactcctgacgtcagtttattttacaccccttaacggcagctgggtgacaaacaaaaaacgacagaaaaccacggtttgataccctccaaacagtggttttctgtcgtccaaaaatagccgaaaagtgttgacgtatacacttgttttcggtaaaatgaagacataacttaaacattgtaagtgagggcttacaaaccaagtgttcgatgctgcaacatcggacactttttatttgtcattctttatttgtattcaattttgcaaatagctcgcaaacaaaatatgtatcatcaaatctattaaccttgttgtctgcaaacaacagggttttttgttgtttatttagaataactagaaccagaattcaatgccaaaactttcacattgacttaacttgactttatcttacacgattttttttttgacgtaaagccccgggcctgaaatcacttttctctactgatttcactgatttcatttttattatataatcctcaaatagcctgtattcactgattttaaatgtgatttcattttattgactttagtgatataagatgctagtattgaggaaagtgaaatcaaaggagagaataaaaatatgaatatttctcgtatgaacgtggactttggaaacagtatgtacatgaatttaattgatggttatattttgaattgcctacaaatgtagtagagatatctaaagaagctgctgaaggaaaatttacgagtatcgttgaagatccggcagatttaaaggaccggttattagtttctacagttattgatgaaacagagagatattttctagttggtgaacttgctgaaccagaagtgttaggcaaccaacacatcaagaagttacataataaagtagagtcacatattccatacgtaacatttttagctgcaactgcttattaccaagcgctaaaaggcaaacgtgaagataatgaagttactattgaatactttcaaacaatgctaccaatttggcttcttaaaaaattggataagttcagtgaaatgcagaaaaggatggcatctaaatttttgggcactcaccaagtaaaggtgctgacattaggattagaaaaagagcttactataaaagtggaagatgcagcgtgcaggatcgaatctgaagtagcaagatgggcaataaagaaaaactttgacctagaagataaagactatgccgaacaatttaaaaattatgacgtagttttttgtgatttaggtggcggaacagatgatctagtattactaccagctggattaaaaccgccaaaaagtcgtgattcttttgtttctaataccgaagcaccgtttttagcgcacttagaaaaattgagaaaagaaaaactcctagagcactttgatagcgttagggagcttgaaaagtttatatactcaaatattggaaaaactaagatggaacgaagagacgggaataccggtcagaaatttgatttaactgatatcatcaaaaaatctcttaaagaatacacagaaatcaaaatagcccaagctgaaaatacgttccctgcaccaaaagataaggtttacaaatacctttattttggcggtgttggcgaggtgcttgaagaatcaattagtgtggttactgaagagagatatggccgtgatatttctgaatcaaatcatatagttgctgaggatgcaagactgctcaacttatatggccttgaagttttaagccgcgctgaacaagtaaagaaacaggcaaatgaaaaagaggcacaatcaatttaggtgattagaaatggggaaaaacaaaagaattccactctttaatgtccgaacaacacaaatgtctgatgaaatgtacgattttgttttagagcagattagtacattcagtaaaggtaagagtaagggtacctttagagagtatgcctttcagctcatagaaagggacatgcaacaacagaaagaggaacagcaaaatagagaaaaagatcgtcatgttcatgatgaattaattgccatgagagaagaaatgaaaaaagaatttcgtgatttgaggaaaaaaattgatcagggatcgatctacgtagaacacaaaacagctgatccaaagtcagcttcaaaaacgattgaagaaggtcagttaatcactgaaaaaatcactggaactattgaagaagaatacgactatgatttttaagagcctggattaatctaggctctttttttatgccatttaagggaggattgcatgacaaactttttttagttgcaacac agacgccctgagcaac Alp7A(SEQ ID NO: 6) atgaatatttctcgtatgaacgtggactttggaaacagtatgtacatgaatttaattgatggttatttttttgaattgcctacaaatgtagtagagatatctaaagaagctgctgaaggaaaatttacgagtatcgttgaagatccggcagatttaaaggaccggttattagtttctacagttattgatgaaacagagagatattactagaggtgaacttgctgaaccagaagtgttaggcaaccaacacatcaagaagttacataataaagtagagtcacatattccatacgtaacatttttagctgcaactgcttattaccaagcgctaaaaggcaaacgtgaagataatgaagttactattgaatactttcaaacaatgctaccaatttggcttcttaaaaaattggataagttcagtgaaatgcagaaaaggatggcatctaaatttttgggcactcaccaagtaaaggtgctgacattaggattagaaaaagagcttactataaaagtggaagatgcagcgtgcaggatcgaatctgaagtagcaagatgggcaataaagaaaaactttgacctagaagataaagactatgccgaacaatttaaaaattatgacgtagttttttgtgatttaggtggcggaacagatgatctagtattactaccagctggattaaaaccgccaaaaagtcgtgattcttttgtttctaataccgaagcaccgtttttagcgcacttagaaaaattgagaaaagaaaaactcctagagcactttgatagcgttagggagcttgaaaagtttatatactcaaatattggaaaaactaagatggaacgaagagacgggaataccggtcagaaatttgatttaactgatatcatcaaaaaatctcttaaagaatacacagaaatcaaaatagcccaagctgaaaatacgttccctgcaccaaaagataaggtttacaaatacctttattttggcggtgttggcgaggtgcttgaagaatcaattagtgtggttactgaagagagatatggccgtgatatttctgaatcaaatcatatagttgctgaggatgcaagactgctcaacttatatggccttgaagttttaagccgcgctgaacaaaaagaaacaggcaaatgaaaaagaggcacaatcaatttag AlD7R (SEQ ID NO: 7)atggggaaaaacaaaagaattccactctttaatgtccgaacaacacaaatgtctgatgaaatgtacgattttgttttagagcagattagtacattcagtaaaggtaagagtaagggtacctttagagagtatgcctttcagctcatagaaagggacatgcaacaacagaaagaggaacagcaaaatagagaaaaagatcgtcatgttcatgatgaattaattgccatgagagaagaaatgaaaaaagaatttcgtgatttgaggaaaaaaattgatcagggatcgatctacgtagaacacaaaacagctgatccaaagtcagcttcaaaaacgattgaagaaggtcagttaatcactgaaaaaatcactggaactattgaagaagaatacga ctatgatttttaaga lacZ(SEQ ID NO: 8) atgatagatcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccattcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggatccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgagatgaaagctggctacaggaaggccagacgcgaattatttagatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggattatgcatcgagctgggtaataagcgaggcaatttaaccgccagtcaggctctctttcacagatgtggattggcgataaaaaccaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgagcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaagcggccgctcgagtctaa T1 terminator (SEQ ID NO: 9)cggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcc tctagcac lacI(SEQ ID NO: 10) atgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggagccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggca gtga plac1q(SEQ ID NO: 11) cctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcacca

This genetic construct was transformed into the E. coli ECN strainthrough the electroporation technique described above in Examples 1 and2.

Example 6: Enzyme-Substrate Reaction Performed in Subject withMetastatic Tumor Identification

Our platform consists of an engineered EcN strain containing aninducible β-galactosidase (lacZ) expression vector, together calledEcN-lacZ (FIG. 2A). The EcN-lacZ system can be remotely switched ON via20 mM IPTG drinking water, enabling rapid growth before high level lacZexpression is initiated. We used two engineered plasmid maintenancesystems (hok and alp7A) to ensure efficient propagation of the lacZexpression vector in vivo. This combination of toxin-antitoxin (hok) anddynamic filament (alp7A) maintenance systems results in a substantialincrease in the number of stable generations at full lacZ expression. Wechromosomally integrated a luciferase expression cassette to monitorEcN-lacZ colonization using our time-lapse in vivo circuitquantification platform (T. Danino et al. ACS Synth. Biol. 2012, 1,465-470). After oral administration of EcN-lacZ cells, one or morecommercially available indicator substrates are injected systemically(FIG. 2A). These substrate(s) circulate throughout the bloodstream anddiffuse into the tumor environment to be cleaved by EcN-lacZ bacteriabefore being collected in the bladder, yielding a measurable indicatorof tumor presence in the urine.

To test whether EcN-lacZ bacteria can colonize small, metastatic tumors,we orally administered EcN-lacZ cells to previously described mousemodels of liver metastasis using an MC26 colorectal cell line. Roughly24 hours following gavage with 3×10⁹ EcN-lacZ bacteria, we excised thelivers and observed bacterial luminescence in tumor patches for micewhere EcN-lacZ bacteria were administered, but not in control tumor mice(FIG. 2B, lower left panels). Notably, the small size of thesemetastatic tumors meant this colonization could not be detected directlyby whole-animal IVIS imaging, as in a traditional diagnostic (FIG. 2B,upper panel comparison). Tumor-specificity was verified by observing alack of colonization in other internal organs (FIG. 2B, bottom rightpanels).

To demonstrate that our EcN-lacZ can quickly and reliably detect thesetumors, we injected LuGal into EcN-lacZ, EcN, and noncolonized mice24-hours after gavage and collected urine samples. We quantified thefraction of cleaved substrate by in vitro luciferase assays and comparedthe EcN-lacZ signal to either EcN or noncolonized signal (FIG. 2C). Weobserved clear statistical differences between the control tumor andEcN-lacZ mice (N=5, p<0.01) and between the EcN and EcN-lacZ mice (N=3,p<0.001). The results demonstrate that 4-fold increase of signal overthe signal acquired by mice treated without tumors, and a 2-fold signalincrease above the signal acquired from tumor background. Tocharacterize instrument-free application, we injected CPRG totumor-bearing and control mice. Upon collection of urine we observed aclear color change for the tumor mouse (FIG. 2D, right panel depictsyellow urine from control mice and left panel depicts red colored urinefrom tumor-bearing mice). A notable advantage of our diagnostic systemis that it integrates the signal from all sources of tumor burden in theurine, even though individual metastases are too small to detect ontheir own by IVIS luminescence imaging (FIG. 2B, top panel). Thus, theEcN-lacZ diagnostic may identify the presence of small metastases soonerthan conventional assays that rely on observing individual tumors. Insome embodiments, the diagnostic may work within 24 hour sofadministration to allow sufficient colonization of bacteria andsecretion of quantities of enzyme sufficient to catalyze a reaction witha substrate whose reaction products enter the urine of a subject indetectable levels.

Our system leverages commercially available substrates to yield aversatile platform with multiple diagnostic modalities, includinginstrument-free methods that rely on color changes. Despite a large bodyof research on blood-based biomarkers for cancer, current diagnosticsstruggle to detect small metastases since poor circulation times. OurEcN-lacZ system combats this problem with exponential growth of thediagnostic agent, enzymatic cleavage, and signal integration in theurine. Since EcN is safe and currently prescribed in humans, thisplatform may function with at-home paper tests, field diagnostics, andintegrated with existing medical infrastructure for urinalysis.

Example 7: Enzyme-Substrate Reaction Performed in Subject withNon-Metastatic Tumor Identification

Mice were fed compositions comprising E. coli Nissile probioticscomprising the nucleic acid sequences comprising. Urine of the animalswith and without solid tumors was collected on 96-well plates, andtested for luminescence. Results are depicted in FIG. 3. Once inside thetumor, the bacteria grow and the colony expands, reaching levels of10⁶-10⁹ bacteria cfu/gram in the first 24-48 hours. This leads to highlevel of amplification of a desired diagnostic product the bacteria willproduce to create the color change. The diagnostic product can be asmall molecule that diffuses into the bladder, a secreted enzyme, or anon-secreted enzyme which can cleave an additional substrate that isintroduced. As a representative example, we used the Beta-galactosidase(lacZ) enzyme. LacZ is an ideal platform enzyme as it is the most highlydeveloped biological reporter with dozens of commercially availablesubstrates. 24 hours following colonization with oral delivery of ourlacZ-expressing strain, we introduce the substrate systemically. Examplesubstrates tested include S-Gal, Ch-Red, and LuGal which each have theirown unique applications. ChRed-Gal, when cleaved produces a smallmolecule that changes to a purple color and can be visually detectablein the urine (FIG. 2b , see mice without tumor (yellow urine or lightgrey if in grey scale) versus tumor-bearing mice that excrete darkcolored urine (or dark grey if in grey scale). S-Gal, when cleaved,produces a small molecule which can be detected in the urine upon Fe3+addition. LuGal, when cleaved, produces a luciferin molecule which canbe sensitively detected in the urine with a luciferase assay. FIG. 3cshows the LuGal based assay for tumor-bearing mice injected with lacZbacteria, tumor-bearing mice without colonized bacteria, and non-tumormice. The signal to noise ratio is approximately 5-10-fold overbackground signals, which makes it straightforward to determine if atumor exists (FIG. 3D). Together, these approaches constitute aversatile diagnosis platform leverages the dozens of commerciallyavailable substrates and existing medical infrastructure for urinalysis(UA).

Example 8: Bacterial Compositions Comprising Hok System and In VivoPlasmid Stability

By increasing the plasmid stability of the plasmids in thenon-pathogenic bacteria, sufficient amounts of enzyme or other proteinsencoded by the nucleic acids in the bacteria can be utilized. Modifiedbacterial strain can become efficient vehicles for either: (i)delivering toxic payloads to tumor cells or cells associated withhyperproliferative disorders or delivering an enzyme whose reactionproduct can be processed at levels sufficient for detection in the urineof a subject treated with the modified bacteria. FIGS. 6 and 7 describean experiment in which ECN bacteria were grown to an OD=0.4-0.8, spundown and washed with PBS 3×, and then injected into nude mice bearingcolorectal tumors (LS174T) of 4-10 mm in size. After 24 or 72 hours,tumors were extracted and homogenized using a Miltenyi dissociator with5 mL of PBS+15% glycerol. Samples were plated at appropriate dilutionsand with 2-3 replicates on media containing antibiotics or noantibiotics. Counting colonies from both plates and calculating theratio yielded a measure of the percent of bacteria within the tumor thatstill maintained the plasmid. N=4-5 tumors for each data point. FIGS. 6and 7 describe the rate of growth and percent of plasmid with plasmidover certain timepoints No stability is a colE1 plasmid without hoksystem or alpA polarization protein; hok is a is a colE1 plasmid withhok/sok containing stable elements, and hok+alpA is a colE1 plasmidcontaining both hok elements and alpA stabilizing elements. The graph ofFIG. 6 show that both hok and hok+alpA strains grow at a similar rate invivo, so the plasmid loss rate is not due to bacteria dividing faster inone group versus another. FIG. 7 shows increased plasmid stability inbacteria when transformed with both AlpA genes and hok system genes andbacteria transformed with just the hok system genes. Bacteria with bothAlpA genes and hok system genes demonstrate more stable plasmidstability over a 72 hour period after injection into mice.

Example 9: Manufacture, Stability, Safety and Functional Analyses

Strains & Plasmids:

Plasmid pTKW106alpA was constructed by adding the 3.5 kb alp7AR cassettefrom the B. subtilis natto plasmid pLS20 to the pTKW106 lacZ expressionvector containing the hok/sok plasmid maintenance system^(1,2). Thealp7AR gene (SEQ ID NO:5) by PCR and added AvrII/NheI restriction sitesusing primers P1-2. For the purpose of cloning the alp7AR cassette intopTKW106, the entire 9 kb pTKW106 was amplified and added a single AvrIIrestriction site using primers P3-4. This PCR product was then digestedwith AvrII and ligated with the AvrII/NheI digested alp7AR insert,producing pTKW106alpA. The lux integrated strain was produced bytransforming EcN bacteria with the p16Slux plasmid 3. The plasmid withno stabilizing element (pTKW106_delhok) was created by PCR by removingthe hok/sok region of pTKW106. The plasmids in FIG. 9A are the pluxplasmid (Danino, et al. 2013), pTD103luxCDABE (inducible by 10 uM AHL),and pTD104luxCDABE (inhibited by(Z—)-4-Bromo-5-(bromomethylene)-2(5H)-furanone at 10 uM). The latterwere constructed by replacing the GFP with lux genes in previous work⁴.

Plasmid Stability and Beta-Galactosidase Activity In Vitro.

Strains of E. coli lacZ deletion mutants (Mach One, Invitrogen) weretransformed with either pTKW106, pTKW106alpA, or pTKW106_delhok.Cultures were diluted into LB media (Sigma-Aldrich) with antibiotics andno IPTG, and grown till an approximate OD=0.1. Cultures were thendiluted 1/100× into deep 96 well-plates (Corning #3600) with 500 uL LBwithout antibiotics and grown for 24 hours. Each 24 hours, cultures werediluted 1/10,000× and grown again in an additional well plate for 24hours. Beta-galactosidase activity was measured using a Tecan i200 platereader by diluting wells 1-10× as needed to obtain a cleavage rateduring the 5-60 min time when readings were obtained. Maximal activitieswere obtained from the linear portion of absorbance curve at 575 nm foreach strain and each day. Each condition was replicated 4 times. Plasmidstability in 9c was calculated by plating on an LB+S-Gal and determiningthe number of white and black colonies¹, with the 0 hours time pointbeing a pre-culture grown with antibiotics. In vitro activities in panel9d are normalized for each strain at the 24 hour time point while inpanel 9f, activities are normalized to the norm 1 mM condition.

Tumor Cell Lines, Animal Models and In Vivo Tests.

Plasmid stability tests were performed in 6 week old nude mice (Taconic)bearing tumors from a colorectal cancer cell line, LS174T (ATCC), andtumors were grown for 1-2 weeks till they reached a size of 5-10 mm. Toobtain measures of plasmid stability in vivo, EcN bacteria with pTKWplasmids were injected intravenously at a dosage of 1e6 bacteria, andtumors were steriley extracted and homogenized using a TissueDissociator (Miltenyi) and plated on both LB and LB with antibiotics toobtain the percent of plasmids with cells or measured for lacZ activityas mentioned above⁵. Enzyme activities of homogenates in vivo wereobtained by the same method as in vitro. The liver metastasis model wasgenerated by intraspenically injecting immunocompetent Balb/c mice(Taconic) with a luciferized, metastatic colorectal cell line (MC26 cellline, Tanabe Lab MGH)^(6,7). Animals were monitored via IVIS (IVIS 200,Calipers) for approximately 20 days. 2-5e⁹ EcN bacteria were gavaged anddrinking water was supplemented with IPTG at 10 mM concentration toinduce lacZ expression. Some mice were euthanized and livers extractedfor quantification and detection of bacterial luminescence via IVIS 200(Calipers).

Urine Diagnostic Assay.

Mice were injected subcutaneously with 1 ml sterile PBS 1.5 hours beforetail vein injection with 100 ul Luga1 [75 ul PBS, 25 ul Luga1 (0.01mg/ul)]. The Luga1 injection was immediately followed by a secondinjection of 1 ml sterile PBS subcutaneously to allow for sufficientvolume of urine to be retrieved. Mice were placed in urine collectiontubes for 1-2 hours until urine appeared. 1 ul of urine was used to testfor luciferin with a luciferase assay kit (Promega QuantiLum rLuciferaseKit) via luminescence in a Berthold Centro LB 960 reader.

Biodistribution/q-PCR Experiments.

Organs were harvested sterilely by using a bead sterilizer for organsand washing in ethanol and then water before extraction. A small tissuesample between 10-100 mg was cut from the organ and DNA was isolatedusing the UltraClean Tissue&Cells DNA Isolation Kit (MoBio, Carlsbad,Calif.). 1 uL of DNA sample was used in a subsequent q-PCR experimentrun according to the manufacturer's parameters (Qiagen, Quantitech SYBRKit) on a Bio-RAD iCycler machine. Control curves were run for each qPCRexperiment. Colony counts were obtained in separate experiments wherewhole organs were excised, homogenized in a Tissue Dissociator(Miltenyi) and plated on erythromycin to detect presence of EcN.Comparisons of colony counting from tumors and q-PCR experiments wereequivalent.

Discussion

The ability of certain strains of bacteria to undergo tumor-specificgrowth has been explored for use as a potential cancer therapy and thegrowing ability to engineer genetic circuits with synthetic biology nowenables the design of novel therapies and diagnostics with programmedbehavior. We develop a simple and noninvasive method for delivering genecircuits to systemic tumors via oral delivery of the probiotic bacteriumE. coli Nissle 1917 (EcN) resulting in execution of EcN's geneticprogram within the tumor microenvironment (FIG. 8). Our platform (PROP,for programmable probiotics) merges the safe and proven deliverycharacteristics of probiotics such as EcN with sophisticated programmedsensing and delivery capabilities drawn from synthetic biology. Toillustrate the potential of this platform to develop next-generationdiagnostics by integrating with existing clinical paradigms, we describean implementation of the platform, PROP-Z, that uses dynamicplasmid-based expression of a reporter enzyme to identify and report thepresence of tumors via urinalysis, one of the most common methods ofmedical diagnosis.

Since synthetic genetic circuits have generally been engineered usingplasmids that enable high signal, low noise, and modularity, we used ourPROP platform to test three different circuit architectures(constitutive, inducible, and quorum sensing) using a subcutaneousxenograft model of colorectal cancer (FIG. 9A). Here, systemicallyadministered PROP bacteria localize in tumors and execute theirprogrammed behavior in the tumor microenvironment following intravenousinjection. Since plasmids are often unstable in nutrient-poor conditionslike the tumor microenvironment⁸, we engineered a dual-maintenancevector to ensure long-term stability for our PROP platform (FIG. 9B).The first maintenance device is an R1-derived toxin-antitoxin systemthat simultaneously produces a toxin (hok) and a short-lived antitoxin(sok), killing the cell in the event of plasmid loss. The second device,alp7, comes from the B. subtilis plasmid pLS20 and produces filamentsthat dynamically push plasmids to the poles, ensuring equal segregationduring cell division⁹. To measure the performance of our maintenancesystems, we used a combination of in vitro and in vivo assays. First, weperformed colony counts (+/−S-Gal) after successive subcultures, andobserved virtually complete plasmid maintenance (FIG. 9C) and preservedenzymatic activity of lacZ (FIG. 9D) for at least 48 hours when thecircuits included both hok/sok and alp7. Since the tumormicroenvironment is characterized by low nutrient levels, pH, and oxygencontent^(10,11), we performed further in vitro experiments under relatedconditions to test the performance of our diagnostic (FIG. 9F,G). Weobserved that PROP-Z function was generally maintained with respect tobacterial growth and lacZ function, in that both measures were mildlyreduced, proportional to the extent of the condition tested.

Since complex selective pressures such as those mediated by an intactimmune system cannot be modeled in vitro, we performed furtherdiagnostic testing in vivo using our subcutaneous cancer model. We foundthat less than 10% of bacteria remained in the tumors after 24 hours ifno stabilization circuits were utilized (FIG. 9E). With the addition ofthe hok/sok system, 75% of the bacteria were retained for the first 24hours, followed by a decline to 45% of the initial bacterial load after72 hours (FIG. 9E). With the combination of hok and alp7, over 96%PROP-Z bacteria remained after 72 hours in vivo (FIG. 9E). Thissubstantial improvement in stability likely results from the combinationof pre- and post-division mechanisms that promote plasmid maintenance,mirroring natural strategies used to achieve this outcome¹². The fullystabilized, high-copy expression vector may also be relevant foradditional applications of synthetic biology in vivo. A notableadvantage of our PROP-Z platform is its capacity for enhancing thesignal to noise ratio via three modes of signal amplification-bacterialgrowth, enzyme production, and enzymatic turnover. While traditionaldiagnostics are limited by the small fraction of the injected dose thatreaches the site of interest¹³, the small number of PROP-Z bacteria thatinitially reach the tumor quickly expand to achieve a maximized signal.In addition, enzymatic turnover further multiplies signal gain sinceeach enzyme can cleave many substrates. To quantify the degree ofamplification achieved by our diagnostic, we excised and homogenizedcolonized subcutaneous tumors over the course of 3 days, performingcolorimetric assays for lacZ activity at each time point. Aftersubtracting background due to nonspecific cleavage, the combined signalamplification of bacterial exponential growth and enzymatic turnoverproduced signals nearly 5-fold greater than the initial injected dose(FIG. 9H). The multiple modes of amplification help generate signalsthat rise above the in vivo background, a significant challenge inpoint-of-care diagnostics¹⁴.

We next sought to apply PROP-Z probiotics in a pre-clinical assay tomeasure its capacity to overcome the clinical challenge of detectingcancer metastases, which are ultimately responsible for 90% of allcancer-related deaths but remain difficult to detect because of theirsmall size and multiplicity¹⁵. The portal venous system flows from theGI tract to the liver, thus, we hypothesized that after oraladministration, probiotics would follow blood flow patterns and directlycolonize liver metastases. To test this prediction, we chose a syngeneicmodel of colorectal cancer liver metastases (FIG. 10A). To directlyvisualize probiotics within the liver metastases, we orally administeredPROP-Z bacteria expressing a chromosomally-integrated luminescencecassette¹⁶. These small doses of bacterial were sufficient to detectluminescent signals in metastases as small as 1 mm, and we observedbacterial infiltration in virtually all tumor cores (FIG. 10B,C). Toquantify the specificity of colonization, we developed a quantitativePCR-based assay to measure PROP-Z bacteria in various organs followingoral administration. Over a period of 7 days, we measured the number ofbacteria in liver metastases and found a striking colonization level of10⁶ bacteria (FIG. 10D). In stark contrast, PROP-Z bacteria infiltrationin control organs (spleen and kidneys) was below the limits of detectionfor our q-PCR assay (<500 bacteria/g). As confirmation of this negativeresult, we assayed for off-target colonization by performing colonycounts of entire organs, and observed zero PROP-Z bacteria in anycontrol organ tested. As a result of zero off-target colonization,PROP-Z treated mice survived without any noticeable adverse effects forat least 9 months. Collectively, these findings constitute the firstdemonstration of GI translocation and specific colonization of livermetastases by oral delivery of the probiotic EcN, setting the stage forchaperoned delivery of gene circuits to systemic tumors via oraldelivery.

What is claimed is:
 1. A composition comprising a non-pathogenicmicroorganism comprising: a first nucleic acid sequence encoding apolarization protein, wherein the first nucleic acid sequence comprisesa nucleic acid sequence having at least 90% sequence identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 6 and SEQ ID NO: 7; and a second nucleic acid sequenceencoding a combination of a toxin and an antidote, wherein the secondnucleic acid sequence comprises a nucleic acid sequence having at least90% sequence identity to SEQ ID NO:
 4. 2. The composition of claim 1,wherein the non-pathogenic microorganism is a bacterium.
 3. Thecomposition of claim 1, wherein the non-pathogenic microorganism is aGram-negative bacterium.
 4. The composition of claim 1, wherein thenon-pathogenic microorganism is Escherichia spp., Firmicutes spp.,Bacteroidetes spp., Lactobacillus spp., Bifidobacteria spp., orAcidopholus spp.
 5. The composition of claim 1, wherein thenon-pathogenic microorganism is E. coli Nissle 1917 (EcN).
 6. Thecomposition of claim 1, wherein the non-pathogenic microorganism isharvested from a sample obtained from a human or animal.
 7. Thecomposition of claim 1, wherein the polarization protein is acytoplasmic microtubule associated protein.
 8. The composition of claim1, wherein the polarization protein is a combination of AlpA and AlpR.9. A kit comprising: (i) the non-pathogenic microorganism composition ofclaim 1; (ii) at least a first container comprising a rehydrationsolution; and, optionally, (iii) a syringe and/or needle.
 10. A foodproduct comprising the nonpathogenic microorganism of claim 1.